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SREA is involved in regulation of siderophore biosynthesis, utilization and uptake in Aspergillus nidulans
Abstract
Under conditions of low iron availability, most fungi excrete siderophores in order to mobilize extracellular iron. We show that lack of the GATA-type transcription factor SREA in Aspergillus nidulans not only leads to derepression of siderophore biosynthesis but also to deregulation of siderophore-bound iron uptake and ornithine esterase expression. Furthermore, SREA deficiency causes increased accumulation of ferricrocin, the siderophore responsible for intracellular iron storage. In sreA deletion strains, extracellular siderophore production is derepressed but still regulated negatively by iron availability, indicating the presence of an additional iron-regulatory mechanism. In contrast, iron affects ferricrocin accumulation in a positive way, suggesting a protective role for this siderophore in detoxification of intracellular iron excess. The harmfulness of deregulated iron uptake in this mutant is demonstrated by increased expression of genes encoding the antioxidative enzymes catalase CATB and the superoxide dismutases SODA and SODB. It is noteworthy that iron starvation was found to repress catB expression in wild-type (wt) and SREA-deficient strains, consistent with catB being subject to SREA-independent iron regulation. Differential display led to the identification of putative SREA target genes amcA and mirA. The deduced MIRA amino acid sequence displays significant similarity to recently characterized siderophore permeases of Saccharomyces cerevisiae. amcA encodes a putative mitochondrial carrier for the siderophore precursor ornithine, indicating cross-regulation of siderophore and ornithine metabolism.
Supplementary resources (3)
Nucleotide Sequence
February 2001
Nucleotide Sequence
February 2001
Nucleotide Sequence
February 2001
... Pseudomonas fluorescens [26] Aspergillus nidulans [29] Catechol salt-type fields. It aims to provide a comprehensive domain of this domain's current research status, development trends, and pressing issues. ...
... Pseudomonas fluorescens [26] Aspergillus nidulans [29] Catechol salt-type Strong lipophilicity and high affinity with Fe; strong resistance to environmental pH changes ...
Siderophores are a class of small molecules renowned for their high iron binding capacity, essential for all life forms requiring iron. This article provides a detailed review of the diverse classifications, and biosynthetic pathways of siderophores, with a particular emphasis on siderophores synthesized via nonribosomal peptide synthetase (NRPS) and non-NRPS pathways. We further explore the secretion mechanisms of siderophores in microbes and plants, and their role in regulating bioavailable iron levels. Beyond biological functions, the applications of siderophores in medicine, agriculture, and environmental sciences are extensively discussed. These applications include biological pest control, disease treatment, ecological pollution remediation, and heavy metal ion removal. Through a comprehensive analysis of the chemical properties and biological activities of siderophores, this paper demonstrates their wide prospects in scientific research and practical applications, while also highlighting current research gaps and potential future directions.
... In agreement with results reported previously by Danion et al. (30), early germ tubes were formed at 6 h, and the germination of all remaining conidia was completed in 10 h (30). In general, A. fumigatus mycelia are grown from an appropriate morphotype (31) (conidial, isotropic growth, or polarized growth state) depending on strain origin, micronutrient availability, temperature, and pH (32). With the inoculum that we used in the in vitro model, specific siderophores secreted at each stage of A. fumigatus growth were measured by HPLC-MS-based infection metallomics. ...
... FsC dominates in the cell during initial hyphal elongation (32) and represents the intracellular precursor of TafC (37). Once TafC amounts are sufficient and fungal need for iron is saturated, SreA-and HapX-mediated iron regulation (38,39) starts to block gene expression involved in TafC synthesis via FsC. ...
The importance of this research lies in the demonstration that siderophore analysis can distinguish between asymptomatic colonization and invasive pulmonary aspergillosis. We found clear associations between phases of fungal development, from conidial germination to the proliferative stage of invasive aspergillosis, and changes in secondary metabolite secretion.
... Formaldehyde agarose gels were used to separate 10 mg of total RNA, which was then blotted onto Amersham Hybond N 1 membranes. Hybridization with the appropriate digoxigenin (DIG)-labeled probes was performed as previously described (66). The primers used for PCR amplification of hybridization probes can be found in Table S3. ...
... Siderophores and radiolabeling of iron-free siderophores. Triacetylfusarinine C and fusarinine C were produced and isolated in-house from iron-starved liquid A. fumigatus cultures as described previously (30,66). The iron-free siderophores TAFC, FsC and FC were labeled for uptake assays as previously described (13,(67)(68)(69), by using 200 mL of a 68 GaCl 3 eluate (;20 to 30 MBq) obtained by the fractionated elution of a 68 Ge/ 68 Ga generator (IGG100; Eckert and Ziegler Isotope Products, Berlin, Germany) with 0.1 M hydrochloric acid. ...
Siderophores play an important role in fungal virulence, serving as trackers for in vivo imaging and as biomarkers of fungal infections. However, siderophore uptake is only partially characterized. As the major cause of aspergillosis, Aspergillus fumigatus is one of the most common airborne fungal pathogens of humans. Here, we demonstrate that this mold species mediates the uptake of iron chelated by the secreted siderophores triacetylfusarinine C (TAFC) and fusarinine C by the major facilitator-type transporters MirB and MirD, respectively. In a murine aspergillosis model, MirB but not MirD was found to be crucial for virulence, indicating that TAFC-mediated uptake plays a dominant role during infection. In the absence of MirB, TAFC becomes inhibitory by decreasing iron availability because the mutant is not able to recognize iron that is chelated by TAFC. MirB-mediated transport was found to tolerate the conjugation of fluorescein isothiocyanate to triacetylfusarinine C, which might aid in the development of siderophore-based antifungals in a Trojan horse approach, particularly as the role of MirB in pathogenicity restrains its mutational inactivation. Taken together, this study identified the first eukaryotic siderophore transporter that is crucial for virulence and elucidated its translational potential as well as its evolutionary conservation.
... In the wild-type expression profile, a GATA transcription factor was significantly up-regulated in spherules but down-regulated in the mutant. This gene is conserved across fungi and implicated in iron homeostasis, siderophore biosynthesis, and morphogenesis (Oberegger et al., 2001;Hwang et al., 2008;Gauthier et al., 2010;Attarian et al., 2018). These data suggest that the acquisition of FIGURE 3 | Venn diagram of differentially expressed genes between mycelia and spherules for each strain, respectively. ...
... Additionally, transcription factors (TFs) such as the highly conserved class of GATA TF's activate alternative nitrogen processing pathways in the absence of preferred sources. These regulators are often highly expressed in both in vitro and in vivo infection conditions and are linked to fungal virulence (Oberegger et al., 2001;Gauthier et al., 2010;Lee et al., 2011;Chung et al., 2012;Hwang et al., 2012). In C. neoformans, the Gat1 TF regulates capsule formation (Lee et al., 2011). ...
Coccidioides immitis and C. posadasii are soil dwelling dimorphic fungi found in North and South America. Inhalation of aerosolized asexual conidia can result in asymptomatic, acute, or chronic respiratory infection. In the United States there are approximately 350,000 new infections per year. The Coccidioides genus is the only known fungal pathogen to make specialized parasitic spherules, which contain endospores that are released into the host upon spherule rupture. The molecular determinants involved in this key step of infection remain largely elusive as 49% of genes are hypothetical with unknown function. An attenuated mutant strain C. posadasii Δcts2/Δard1/Δcts3 in which chitinase genes 2 and 3 were deleted was previously created for vaccine development. This strain does not complete endospore development, which prevents completion of the parasitic lifecycle. We sought to identify pathways active in the wild-type strain during spherule remodeling and endospore formation that have been affected by gene deletion in the mutant. We compared the transcriptome and volatile metabolome of the mutant Δcts2/Δard1/Δcts3 to the wild-type C735. First, the global transcriptome was compared for both isolates using RNA sequencing. The raw reads were aligned to the reference genome using TOPHAT2 and analyzed using the Cufflinks package. Genes of interest were screened in an in vivo model using NanoString technology. Using solid phase microextraction (SPME) and comprehensive two-dimensional gas chromatography – time-of-flight mass spectrometry (GC × GC-TOFMS) volatile organic compounds (VOCs) were collected and analyzed. Our RNA-Seq analyses reveal approximately 280 significantly differentially regulated transcripts that are either absent or show opposite expression patterns in the mutant compared to the parent strain. This suggests that these genes are tied to networks impacted by deletion and may be critical for endospore development and/or spherule rupture in the wild-type strain. Of these genes, 14 were specific to the Coccidioides genus. We also found that the wild-type and mutant strains differed significantly in their production versus consumption of metabolites, with the mutant displaying increased nutrient scavenging. Overall, our results provide the first targeted list of key genes that are active during endospore formation and demonstrate that this approach can define targets for functional assays in future studies.
... However, the SreA ortholog (Cir1) of Cryptococcus neoformans is required for capsule formation and full virulence in mice (Jung et al., 2006). SreA orthologs have also been characterized to act as an iron repressor in A. nidulans (also called Emericella nidulans) (Haas et al., 1999;Oberegger et al., 2001), Schizosaccharomyces pombe (Pelletier et al., 2002), Penicillium chrysogenum (Haas et al., 1997), Neurospora crassa (Zhou et al., 1998), Histoplasma capsulatum (Chao et al., 2008), and Ustilago maydis . Studies in the grass endophyte Epichloe festucae reveal an important role of SreA-mediated iron regulation in maintaining symbioses with its host Loium perenne (Forester et al., 2019). ...
... Although similar growth reduction resulting from sreA deletion has been reported in C. heterostrophus reducing by 34% (Zhang et al., 2013), it seems that loss of sreA has more drastic effects on the growth of A. alternata than other fungi. Mutation of the sreA homolog in A. fumigatus, A. nidulans, U. maydis, or S. pombe results in little or no growth reduction Haas et al., 1999;Oberegger et al., 2001;Pelletier et al., 2002;Schrettl et al., 2008). In A. alternata, growth deficiency in ΔsreA can be restored nearly to the wildtype level by adding the iron chelator BPS into medium, indicating that severe growth reduction of ΔsreA is mainly attributed to iron toxicity and that SreA negatively regulates iron uptake to avoid excessive iron toxicity. ...
The siderophore-mediated iron uptake machinery is required by the tangerine pathotype of Alternaria alternata to colonize host plants. The present study reports the functions of the GATA-type transcription regulator SreA by analyzing loss- and gain-of-function mutants. The expression of sreA is transiently upregulated by excess iron. The sreA deficient mutant (ΔsreA) shows severe growth defect but produces ACT toxin and incites necrotic lesions on citrus leaves as efficiently as wild type. SreA suppresses the expression of genes encoding polypeptides required for siderophore biosynthesis and transport under iron-replete conditions. Under iron-replete conditions, SreA impacts the expression of the genes encoding the NADPH oxidase complex involved in H2O2 production. SreA negatively impacts H2O2 resistance as ΔsreA increases resistance to H2O2. However, sreA deficiency has no effects on the expression of genes encoding several key factors (Yap1, Hog1, and Skn7) involved in oxidative stress resistance. ΔsreA increases resistance to calcofluor white and Congo red, which may suggest a role of SreA in the maintenance of cell wall integrity. Those are novel phenotypes associated with fungal sreA. Overall, our results indicate that SreA is required to protect fungal cells from cytotoxicity caused by excess iron. The results also highlight the regulatory functions of SreA and provide insights into the critical role of siderophore-mediated iron homeostasis in resistance to oxidative stress in A. alternata.
... In bacteria, these hydrophilic siderophores are composed of acylated and hydroxylated alkylamines, such as the siderophore pyoverdine; produced by Pseudomonas fluorescens, while in fungi, they are composed of hydroxylated and alkylated ornithine [6], for example, trichoderma spp produces fecal coprogens. Except for the siderophore fusarinine C containing ester bond produced by Aspergillus fumigatus, other hydroxamate siderophores contain peptide chains [7]. A bidentate ligand is formed between the two oxygen molecules of the hydroxyformic acid group on the hydroxamic acid hydroxamate siderophores and iron. ...
... In mammals, intracellular iron homeostasis is mainly contr olled thr ough the coordinated posttranscriptional regulation of di v erse iron metabolism genes ( 18 ). In filamentous fungi, iron homeostasis is primarily maintained via transcriptional regula tion media ted by two transcription factors, including the bZIP transcription factor HapX ( h eme a ctivator p rotein X) and the GATA zinc finger transcription factor SreA ( s ideropho re transcription factor A) (19)(20)(21)(22). The roles of HapX and SreA in iron homeostasis have been well characterized in A. fumigatus . ...
Plant pathogens are challenged by host-derived iron starvation or excess during infection, but the mechanism through which pathogens counteract iron stress is unclear. Here, we found that Fusarium graminearum encounters iron excess during the colonization of wheat heads. Deletion of heme activator protein X (FgHapX), siderophore transcription factor A (FgSreA) or both attenuated virulence. Further, we found that FgHapX activates iron storage under iron excess by promoting histone H2B deubiquitination (H2B deub1) at the promoter of the responsible gene. Meanwhile, FgSreA is shown to inhibit genes mediating iron acquisition during iron excess by facilitating the deposition of histone variant H2A.Z and histone 3 lysine 27 trimethylation (H3K27 me3) at the first nucleosome after the transcription start site. In addition, the monothiol glutaredoxin FgGrx4 is responsible for iron sensing and control of the transcriptional activity of FgHapX and FgSreA via modulation of their enrichment at target genes and recruitment of epigenetic regulators, respectively. Taken together, our findings elucidated the molecular mechanisms for adaptation to iron excess mediated by FgHapX and FgSreA during infection in F. graminearum and provide novel insights into regulation of iron homeostasis at the chromatin level in eukaryotes.
... FeCl 3 was added to the filtrate to a final concentration of 1.5mM to convert de-ferri siderophores to the ferri forms. These ferri forms were purified by using amberlite XAD-16 resin (CWG) according to Oberegger et al. (2001) and were eluted with methanol. Fractions were collected at equal volumes (1mL for each fraction) and extracellular siderophores were measured photometrically at 435nm. ...
Hydroxamate siderophores are low molecular weight, high-affinity ferric iron-chelating compounds which are excreted by Aspergillus fumigatus for iron transport extracellularly or iron storage intracellularly. Aspergillus fumigatus was isolated from different samples, then siderophores were extracted from A. fumigatus by phenol, and diethyl ether, and purified by amberlite XAD-16 resin. The chemical structure of siderophores was described by H¹NMR,ESI-MS/MS, andLC-MS/MS analysis. Also, antimicrobial activity was observed. From the elution profile of siderophores, the major peak was at fraction 28. As a result of H¹NMR analysis, the singlets assigned to the -C=O-CH=C (CH3) C-substructure were detected. The siderophore TafC had antibacterial activity against both G +ve and G -ve bacteria. Under different concentrations of iron, it can be deduced that the concentration of 0.03 mmol of iron at 20 hrs. incubation period was an ideal condition for mycelial fresh weight which positively affected, so production of siderophores increased. From chemical analysis, it was found that the chemical formula C39H58FeN6O15 was consistent with the known siderophore N, N’, N”-triacetylfusarinine C (TafC).
... Majority of fungi possess ability to take up iron brought by xenosiderophores, also called heterologous siderophores (siderophores synthesised by other microbial communities). For example, Aspergillus nidulans exhibiting affinity for ferrirubin released from Aspergillus ochraceus (Oberegger et al. 2001) and for bacterially synthesised catechol, enterobactin (Fiedler et al. 2001). Hence, a chance for competition may persist between microorganisms for iron sequestration. ...
Iron is one of the highly abundant elements on the earth’s crust, an essential micronutrient for a majority of life forms, and exists in two frequent oxidation states such as ferrous (Fe²⁺) and ferric (Fe³⁺). These two oxidation states are interconvertible by redox reactions and form complexes with a wide range of siderophores. At neutral pH in soil, Fe²⁺ is highly soluble upto 100 mM but have less biological value, whereas Fe³⁺ is less soluble upto 10–9 M. This reduced bioavailability of Fe³⁺ induces competition among microorganisms. As many microorganisms need at least 10–6 M of Fe³⁺ form of iron for their growth, siderophores from these microbes readily withdraw Fe³⁺ iron from a variety of habitats for their survival. In this review, we bring into light the several recent investigations related to diverse chemistry of microbial siderophores, mechanisms of siderophore uptake, biosynthetic gene clusters in microbial genomes, various sources of heavy metal cations in soil, siderophore-binding protein receptors and commercialisation perspectives of siderophores. Besides, this review unearths the recent advancements in the characterisation of novel siderophores and its heavy metal complexes alongside the interaction kinetics with receptors.
... Ferrioxamine B was purchased from Sigma (Burlington, MA, USA), and ferrioxamine E was purchased from EMC Microcollections GmbH (Tübingen, Germany). The siderophores TAFC and FC used in this study were produced and purified in the laboratory as described by Schrettl et al. and Oberegger et al. (9,44). ...
The opportunistic fungal pathogen Aspergillus fumigatus utilizes two high-affinity iron uptake mechanisms, termed reductive iron assimilation (RIA) and siderophore-mediated iron acquisition (SIA). The latter has been shown to be crucial for virulence of this fungus and is a target for development of novel strategies for diagnosis and treatment of fungal infections. So far, research on SIA in this mold focused mainly on the hyphal stage, revealing the importance of extracellular fusarinine-type siderophores in iron acquisition as well as of the siderophore ferricrocin in intracellular iron handling. The current study aimed to characterize iron acquisition during germination. High expression of genes involved in biosynthesis and uptake of ferricrocin in conidia and during germination, independent of iron availability, suggested a role of ferricrocin in iron acquisition during germination. In agreement, (i) bioassays indicated secretion of ferricrocin during growth on solid media during both iron sufficiency and limitation, (ii) ferricrocin was identified in the supernatant of conidia germinating in liquid media during both iron sufficiency and limitation, (iii) in contrast to mutants lacking all siderophores, mutants synthesizing ferricrocin but lacking fusarinine-type siderophores were able to grow under iron limitation in the absence of RIA, and (iv) genetic inactivation of the ferricrocin transporter Sit1 decreased germination in the absence of RIA. Taken together, this study revealed that ferricrocin has not only an intracellular role but also functions as an extracellular siderophore to support iron acquisition. The iron availability-independent ferricrocin secretion and uptake during early germination indicate developmental, rather than iron regulation. IMPORTANCE Aspergillus fumigatus is one of the most common airborne fungal pathogens for humans. Low-molecular-mass iron chelators, termed siderophores, have been shown to play a central role in iron homeostasis and, consequently, virulence of this mold. Previous studies demonstrated the crucial role of secreted fusarinine-type siderophores, such as triacetylfusarinine C, in iron acquisition, as well as of the ferrichrome-type siderophore ferricrocin in intracellular iron storage and transport. Here, we demonstrate that ferricrocin is also secreted to mediate iron acquisition during germination together with reductive iron assimilation. During early germination, ferricrocin secretion and uptake were not repressed by iron availability, indicating developmental regulation of this iron acquisition system in this growth phase.
... To the separated PCI phase containing the intracellular siderophore, 1 volume water and 5 volumes diethyl ether were added. The intracellular siderophore content of the aqueous phase was measured photometrically at 440 nm using the molar extinction factor 2,460 M 21 cm 21 (14,50). Northern blot analysis. ...
Iron acquisition is crucial for virulence of the human pathogen Aspergillus fumigatus. Previous studies indicated that this mold regulates iron uptake via both siderophores and reductive iron assimilation by the GATA factor SreA and the SREBP regulator SrbA. Here, characterization of loss of function as well as hyperactive alleles revealed that transcriptional activation of iron uptake depends additionally on the Zn2Cys6 regulator AtrR, most likely via cooperation with SrbA. Mutational analysis of the promoter of the iron permease-encoding ftrA gene identified a 210-bp sequence, which is both essential and sufficient to impart iron regulation. Further studies located functional sequences, densely packed within 75 bp, that largely resemble binding motifs for SrbA, SreA, and AtrR. The latter, confirmed by chromatin immunoprecipitation (ChIP) analysis, is the first one not fully matching the 5′-CGGN12CCG-3′ consensus sequence. The results presented here emphasize for the first time the direct involvement of SrbA, AtrR, and SreA in iron regulation. The essential role of both AtrR and SrbA in activation of iron acquisition underlines the coordination of iron homeostasis with biosynthesis of ergosterol and heme as well as adaptation to hypoxia. The rationale is most likely the iron dependence of these pathways along with the enzymatic link of biosynthesis of ergosterol and siderophores.
... They can be produced by bacteria, fungi, and plants [40]. Among the most studied fungi for siderophore production are Aspergillus fumigatus and Aspergillus nidulans, which have 55 similar types of siderophores [41,42]. In addition, Penicillium produces siderophores, capable of solubilizing tricalcium phosphate, even in contaminated soils, opening new opportunities in the field of phytoremediation and agrobiotechnology [43]. ...
Nematophagous fungi (NF) are a group of diverse fungal genera that benefit plants. The aim of this review is to increase comprehension about the importance of nematophagous fungi and their role in phosphorus solubilization to favor its uptake in agricultural ecosystems. They use different mechanisms, such as acidification in the medium, organic acids production, and the secretion of enzymes and metabolites that promote the bioavailability of phosphorus for plants. This study summarizes the processes of solubilization, in addition to the mechanisms of action and use of NF on crops, evidencing the need to include innovative alternatives for the implementation of microbial resources in management plans. In addition, it provides information to help understand the effect of NF to make phosphorus available for plants, showing how these biological means promote phosphorus uptake, thus improving productivity and yield.
... Aspergillus species possess six GATA-type transcription factors termed AreA, AreB, SreA, LreA, LreB and NsdD. AreA and AreB mediate regulation of nitrogen and carbon metabolism (Chudzicka-Ormaniec et al., 2019; Haas et al., 1997), SreA controls iron acquisition (Oberegger et al., 2001;Schrettl et al., 2008), LreA and LreB allow light response (Purschwitz et al., 2008) and NsdD coordinates sexual and asexual development (Lee et al., 2014). Moreover, pxylP contains five putative CreA binding sites outside of the 91bpDS copies. ...
Conditional promoters allowing both induction and silencing of gene expression are indispensable for basic and applied research. The xylP promoter (pxylP) from Penicillium chrysogenum was demonstrated to function in various mold species including Aspergillus fumigatus. pxylP allows high induction by xylan or its degradation product xylose with low basal activity in the absence of an inducer. Here we structurally characterized and engineered pxylP in A. fumigatus to optimize its application. Mutational analysis demonstrated the importance of the putative TATA-box and a pyrimidine-rich region in the core promoter, both copies of a largely duplicated 91-bp sequence (91bpDS), as well as putative binding sites for the transcription factor XlnR and a GATA motif within the 91bpDS. In agreement, pxylP activity was found to depend on XlnR, while glucose repression appeared to be indirect. Truncation of the originally used 1643-bp promoter fragment to 725 bp largely preserved the promoter activity and the regulatory pattern. Integration of a third 91bpDS significantly increased promoter activity particularly under low inducer concentrations. Truncation of pxylP to 199 bp demonstrated that the upstream region including the 91bpDSs mediates not only inducer-dependent activation but also repression in the absence of inducer. Remarkably, the 1579-bp pxylP was found to act bi-bidirectionally with a similar regulatory pattern by driving expression of the upstream-located arabinofuranosidase gene. The latter opens the possibility of dual bidirectional use of pxylP. Comparison with a doxycycline-inducible TetOn system revealed a significantly higher dynamic range of pxylP. Taken together, this study identified functional elements of pxylP and opened new methodological opportunities for its application.
... However, the growth rate was recovered when the organism was grown on the iron-sufficient PDA medium (approximately 20 µM). Similarly, deletion of the sreA homolog in other fungi; Aspergillus fumigatus, A. nidulans, Aureobasidium pullulans, Ustilago maydis, and Shizosaccharomyces pombe resulted in little or no growth reduction [13,14,[34][35][36][37]. In contrast, ∆sreA mutant in Alternaria alternata showed reduced growth both on minimal medium and PDA, but the growth reduction was more prominent on PDA [15]. ...
Siderophores are compounds with low molecular weight with a high affinity and specificity for ferric iron, which is produced by bacteria and fungi. Fungal siderophores have been characterized and their feasibility for clinical applications has been investigated. Fungi may be limited in slow growth and low siderophore production; however, they have advantages of high diversity and affinity. Hence, the purpose of this study was to generate a genetically modified strain in Talaromyces marneffei that enhanced siderophore production and to identify the characteristics of siderophore to guide its medical application. SreA is a transcription factor that negatively controls iron acquisition mechanisms. Therefore, we deleted the sreA gene to enhance the siderophore production and found that the null mutant of sreA (∆sreA) produced a high amount of extracellular siderophores. The produced siderophore was characterized using HPLC-MS, HPLC-DAD, FTIR, and 1 Hand 13 C-NMR techniques and identified as a coprogen B. The compound showed a powerful iron-binding activity and could reduce labile iron pool levels in iron-loaded hepatocellular carcinoma (Huh7) cells. In addition, the coprogen B showed no toxicity to the Huh7 cells, demonstrating its potential to serve as an ideal iron chelator. Moreover, it inhibits the growth of Candida albicans and Escherichia coli in a dose-dependent manner. Thus, we have generated the siderophore-enhancing strain of T. marneffei, and the coprogen B isolated from this strain could be useful in the development of a new iron-chelating agent or other medical applications.
... In many cases the signal triggering transcriptional activation of the biosynthetic genes is connected to nutrient starvation or the onset of reproductive development (Nemeth et al., 2016;Tag et al., 2000). But there are other examples in which rich nutrient sources and active growth is correlated with the production of SMs, such as for the production of siderophores that are required for iron acquisition and metabolism (Oberegger et al., 2001). ...
Co‐culturing the bacterium Streptomyces rapamycinicus and the ascomycete Aspergillus nidulans has previously been shown to trigger the production of orsellinic acid (ORS) and its derivates in the fungal cells. Based on these studies it was assumed that direct physical contact is a prerequisite for the metabolic reaction that involves a fungal amino acid starvation response and activating chromatin modifications at the biosynthetic gene cluster (BGC). Here we show that not physical contact, but a guanidine containing macrolide, named polaramycin B, triggers the response. The substance is produced constitutively by the bacterium and above a certain concentration, provokes the production of ORS. In addition, several other secondary metabolites were induced by polaramycin B. Our genome‐wide transcriptome analysis showed that polaramycin B treatment causes downregulation of fungal genes necessary for membrane stability, general metabolism and growth. A compensatory genetic response can be observed in the fungus that included upregulation of BGCs and genes necessary for ribosome biogenesis, translation and membrane stability. Our work discovered a novel chemical communication, in which the antifungal bacterial metabolite polaramycin B leads to the production of antibacterial defence chemicals and to the upregulation of genes necessary to compensate for the cellular damage caused by polaramycin B.
... While it is true that ROS resistance allows for better competition for nutrients in the wound by BCAs, by contrast, iron allows BCAs to better cope with ROS, because the catalase enzyme is known to require iron for ROS detoxification [104,207]. ...
The use of synthetic fungicides to control fungal diseases has growing limitations due to eco-toxicological risks. Therefore, it is necessary to replace or integrate high risk chemicals with safer tools for human health and environment. Consequently, research on the selection, evaluation, characterization, and use of biocontrol agents (BCAs) has consistently increased in the last decades. BCA formulates, particularly in some countries, are still scarce in coping with the growing demand for their use in sustainable agricultural management. To foster development and utilization of new effective bioformulates, there is a need to optimize BCA activity, to share knowledge on their formulation processes and to simplify the registration procedures. Studies based on new molecular tools can significantly contribute to achieve such objectives. The present review provides the state of the art on biocontrol of fungal plant diseases with special emphasis on (i) features of the most studied BCAs; (ii) key strategies to optimize selection and use of BCAs (iii); mechanisms of action of the main BCAs; (iv) molecular tools and metagenomic studies in the selection and use of BCAs; (v) main issues and constraints in the registration and commercialization of BCAs, and (vi) perspectives in the biocontrol of fungal plant diseases.
... Meanwhile, the toxicity of the SS from M. lusitanicus depended on low iron concentrations in the culture, which positively correlated with rfs mRNA upregulation. Siderophores are produced under low iron concentrations in the medium 39 , and their production is modulated by carbon or nitrogen sources or chemical or www.nature.com/scientificreports/ physical factors, such as pH or temperature 40,41 . ...
Mucormycosis is a fungal infection caused by Mucorales, with a high mortality rate. However, only a few virulence factors have been described in these organisms. This study showed that deletion of rfs, which encodes the enzyme for the biosynthesis of rhizoferrin, a siderophore, in Mucor lusitanicus, led to a lower virulence in diabetic mice and nematodes. Upregulation of rfs correlated with the increased toxicity of the cell-free supernatants of the culture broth (SS) obtained under growing conditions that favor oxidative metabolism, such as low glucose levels or the presence of H2O2 in the culture, suggesting that oxidative metabolism enhances virulence through rhizoferrin production. Meanwhile, growing M. lusitanicus in the presence of potassium cyanide, N-acetylcysteine, a higher concentration of glucose, or exogenous cAMP, or the deletion of the gene encoding the regulatory subunit of PKA (pkaR1), correlated with a decrease in the toxicity of SS, downregulation of rfs, and reduction in rhizoferrin production. These observations indicate the involvement of the cAMP-PKA pathway in the regulation of rhizoferrin production and virulence in M. lusitanicus. Moreover, rfs upregulation was observed upon macrophage interaction or during infection with spores in mice, suggesting a pivotal role of rfs in M. lusitanicus infection.
... Fungal species mostly regulate iron acquisition via Iron Responsive GATA Factors (IRGFs) and the bZIPtranscription factor HapX. When iron is plentiful, IRGFs inhibit transcription of genes responsible of the siderophore system and reductive iron assimilation [58][59][60][61]. Iron starvation induces down-regulation of iron consuming pathways and upregulation of siderophore systems, for example, in A. nidulans via physical interaction between the (nucleotide sequence) CCAAT-binding complex and HapX [62,63]. ...
La résistance aux antibiotiques devient peu à peu un problème majeur de santé publique. La recherche de nouvelles stratégies pour contrer ce phénomène est donc extrêmement importante. Le Fe(III), qui possède un rôle prédominant dans de nombreuses réactions métaboliques, est un nutriment minéral essentiel à la survie des micro-organismes. Pour acquérir cet élément dans les milieux biologiques appauvris en Fe(III), les bactéries ont développé différentes méthodes. L'une de ces stratégies consiste en la synthèse de sidérophores, des molécules chélatrices du Fe(III) de faible poids moléculaire et reconnues spécifiquement par des récepteurs présents sur les membranes bactériennes. Dans la littérature, le développement d'antibiothérapie utilisant la voie des sidérophores via la méthode du « Cheval de Troie » fait l'objet d'une attention particulière. Cette stratégie consiste en la formation d'un conjugué sidérophore-antibiotique permettant la vectorisation de molécules antibactériennes. L'objectif principal de cette thèse est de synthétiser l'acide rhodotorulique (un sidérophore fongique reconnu par des bactéries à Gram négatif telles que E. coli) et des analogues 3,6-disubstitués par des fonctions chélatrices du fer. Pour ce faire, plusieurs voies de synthèses ont été développées : i) des voies reposant sur l'alkylation diastéréosélective de dioxopipérazines portant des copules chirales et ii) des voies de synthèse à partir d'acides aminés modifiés tels que l'acide glutamique ou l'ornithine. En parallèle, la synthèse d'analogues pipéraziniques, plus simples à obtenir, a également été étudiée afin de nous guider quant aux choix des fonctions chélatrices du fer. Des études de reconnaissance, par les bactéries, des analogues obtenus ont été réalisées et la mesure du pouvoir bactéricide de complexes au gallium a été déterminée. Par la suite, les analogues obtenus pourront être couplés à des antibiotiques afin de pouvoir les vectoriser et lutter contre les phénomènes de résistance
... As previously reported in filamentous fungi Aspergillus spp. and Alternaria alternata, two transcription factors SreA and HapX serve as key regulators for fungal adaption to iron excess (59)(60)(61)(62). Therefore, we generated FgHAPX and FgSREA deletion mutants, and determined their sensitivity to high-iron stress at different pH levels. ...
Poaceae plants can locally accumulate iron to suppress pathogen infection. It remains unknown how pathogens overcome host-derived iron stress during their successful infections. Here, we report that Fusarium graminearum (Fg), a destructive fungal pathogen of cereal crops, is challenged by host-derived high-iron stress. Fg infection induces host alkalinization, and the pH-dependent transcription factor FgPacC undergoes a proteolytic cleavage into the functional isoform named FgPacC30 under alkaline host environment. Subsequently FgPacC30 binds to a GCCAR(R = A/G)G element at the promoters of the genes involved in iron uptake and inhibits their expression, leading to adaption of Fg to high-iron stress. Mechanistically, FgPacC30 binds to FgGcn5 protein, a catalytic subunit of Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex, leading to deregulation of histone acetylation at H3K18 and H2BK11, and repression of iron uptake genes. Moreover, we identified a protein kinase FgHal4, which is highly induced by extracellular high-iron stress and protects FgPacC30 against 26S proteasome-dependent degradation by promoting FgPacC30 phosphorylation at Ser2. Collectively, this study uncovers a novel inhibitory mechanism of the SAGA complex by a transcription factor that enables a fungal pathogen to adapt to dynamic microenvironments during infection.
... In many cases the signal triggering transcriptional activation of the biosynthetic genes is connected to nutrient starvation or the onset of reproductive development (Nemeth et al., 2016;Tag et al., 2000). But there are other examples in which rich nutrient sources and active growth is correlated with the production of SMs, such as for the production of siderophores that are required for iron acquisition and metabolism (Oberegger et al., 2001). ...
Direct physical interaction between the bacterium Streptomyces rapamycinicus and the ascomycete Aspergillus nidulans has previously been shown to trigger the production of orsellinic acid and its derivates in the fungus. Here we identified the substance produced by the bacterium that is responsible for this reaction, a guanidine containing macrolide named Polaramycin B. This metabolite is produced constitutively by the bacterium and freely diffuses in an aqueous environment. Above a certain concentration it provokes the production of orsellinic acid without the need of direct physical interaction between the two species. In addition to orsellinic acid the production of several other secondary metabolites was triggered by Polaramycin B. Our genome-wide transcriptome analysis showed that Polaramycin B treatment causes down-regulation of fungal genes necessary for membrane stability as well as genes involved in general metabolism and growth. A compensatory genetic response can be observed in the fungus that included up-regulation of biosynthetic gene clusters and genes necessary for ribosome biogenesis, translation and membrane stability. Our work discovered a specific chemical communication in which Polaromycin B, an antifungal metabolite constitutively produced by different Streptomycete strains, leads to the production of fungal defence chemicals and to the up-regulation of genes necessary to compensate for the cellular damage caused by Polaramycin B.
... In fungi, they consist of hydroxylated and alkylated ornithine amino acid, while in bacteria, they are acylated and hydroxylated alkylamines [37], represented by N 6 -acyl-N 6 -Hydroxylysine or N 5 -acyl-N 5 -Hydroxyornithine reported by Winkelmann [38]. All hydroxamate siderophores are characterized by peptide linkage [24], except fusarinine C (FsC), synthesized by Aspergillus nidulans, which shows ester bonds. Two O2 molecules of these groups bind with Fe, known as bi-dentate ligand. ...
Iron (Fe) is the fourth most abundant element on earth and represents an essential nutrient for life. As a fundamental mineral element for cell growth and development, iron is available for uptake as ferric ions, which are usually oxidized into complex oxyhydroxide polymers, insoluble under aerobic conditions. In these conditions, the bioavailability of iron is dramatically reduced. As a result, microorganisms face problems of iron acquisition, especially under low concentrations of this element. However, some microbes have evolved mechanisms for obtaining ferric irons from the extracellular medium or environment by forming small molecules often regarded as siderophores. Siderophores are high affinity iron-binding molecules produced by a repertoire of proteins found in the cytoplasm of cyanobacteria, bacteria, fungi, and plants. Common groups of siderophores include hydroxamates, catecholates, carboxylates, and hydroximates. The hydroxamate siderophores are commonly synthesized by fungi. L-ornithine is a biosynthetic precursor of siderophores, which is synthesized from multimodular large enzyme complexes through non-ribosomal peptide synthetases (NRPSs), while siderophore-Fe chelators cell wall mannoproteins (FIT1, FIT2, and FIT3) help the retention of siderophores. S. cerevisiae, for example, can express these proteins in two genetically separate systems (reductive and nonreductive) in the plasma membrane. These proteins can convert Fe (III) into Fe (II) by a ferrous-specific metalloreductase enzyme complex and flavin reductases (FREs). However, regulation of the siderophore through Fur Box protein on the DNA promoter region and its activation or repression depend primarily on the Fe availability in the external medium. Siderophores are essential due to their wide range of applications in biotechnology, medicine, bioremediation of heavy metal polluted environments, biocontrol of plant pathogens, and plant growth enhancement.
... Basic Protocol 4 details extracting Nickles et al. Blatzer et al., 2011;Diekmann & Krezdorn, 1975;Konetschny-Rapp et al., 1988;Oberegger, Schoeser, Zadra, Abt, & Haas, 2001 Fumagillin KACC 41191 ...
Fungal secondary metabolites (SMs) have captured the interest of natural products researchers in academia and industry for decades. In recent years, the high rediscovery rate of previously characterized metabolites is making it increasingly difficult to uncover novel compounds. Additionally, the vast majority of fungal SMs reside in genetically intractable fungi or are silent under normal laboratory conditions in genetically tractable fungi. The fungal natural products community has broadly overcome these barriers by altering the physical growth conditions of the fungus and heterologous/homologous expression of biosynthetic gene cluster regulators or proteins. The protocols described here summarize vital methodologies needed when researching SM production in fungi. We also summarize the growth conditions, genetic backgrounds, and extraction protocols for every published SM in Aspergillus fumigatus , enabling readers to easily replicate the production of previously characterized SMs. Readers will also be equipped with the tools for developing their own strategy for expressing and extracting SMs from their given fungus or a suitable heterologous model system. © 2021 Wiley Periodicals LLC.
Basic Protocol 1 : Making glycerol stocks from spore suspensions
Alternate Protocol 1 : Creating glycerol stocks from non‐sporulating filamentous fungi
Basic Protocol 2 : Activating spore‐suspension glycerol stocks
Basic Protocol 3 : Extracting secondary metabolites from Aspergillus spp grown on solid medium
Alternate Protocol 2 : Extracting secondary metabolites from Aspergillus spp using ethyl acetate
Alternate Protocol 3 : High‐volume metabolite extraction using ethyl acetate
Alternate Protocol 4 : Extracting secondary metabolites from Aspergillus spp in liquid medium
Support Protocol : Creating an overlay culture
Basic Protocol 4 : Extracting DNA from filamentous fungi
Basic Protocol 5 : Creating a DNA construct with double‐joint PCR
Alternate Protocol 5 : Creating a DNA construct with yeast recombineering
Basic Protocol 6 : Transformation of Aspergillus spp
Basic Protocol 7 : Co‐culturing fungi and bacteria for extraction of secondary metabolites
... other than A. nidulans have been reported to acquire iron from xenosiderophores (Wiebe and Winkelmann, 1975). Oberegger et al. (2001) have reported uptake of iron via the ferri-enterobactin complex in A. nidulans. Iron is well known to cause oxidative stress via Fenton reaction leading to protein and DNA damage (Yu et al., 2007). ...
Microorganisms produce various secondary metabolites for growth and survival. During iron stress, they produce secondary metabolites termed siderophores. In the current investigation, antifungal activity of catecholate siderophore produced by Escherichia coli has been assessed against Aspergillus nidulans. Exogenous application of the bacterial siderophore to fungal cultures resulted in decreased colony size, increased filament length, and changes in hyphal branching pattern. Growth inhibition was accompanied with increased intracellular iron content. Scanning electron microscopy revealed dose-dependent alteration in fungal morphology. Fluorescent staining by propidium iodide revealed cell death in concert with growth inhibition with increasing siderophore concentration. Antioxidative enzyme activity was also compromised with significant increase in catalase activity and decrease in ascorbate peroxidase activity. Siderophore-treated cultures showed increased accumulation of reactive oxygen species as observed by fluorescence microscopy and enhanced membrane damage in terms of malondialdehyde content. Antifungal property might thus be attributed to xenosiderophore-mediated iron uptake leading to cell death. STRING analysis showed interaction of MirB (involved in transport of hydroxamate siderophore) and MirA (involved in transport of catecholate siderophore), confirming the possibility of uptake of iron–xenosiderophore complex through fungal transporters. MirA structure was modeled and validated with 95% residues occurring in the allowed region. In silico analysis revealed MirA–Enterobactin–Fe3+ complex formation. Thus, the present study reveals a promising antifungal agent in the form of catecholate siderophore and supports involvement of MirA fungal receptors in xenosiderophore uptake.
... All other siderophores were purchased from EMC Microcollections, Germany. Triacetylfusarinine C, fusarinine C, and ferricrocin were produced and isolated in-house from iron-starved A. fumigatus liquid cultures as described previously [38,39]. ...
Siderophore-mediated acquisition of iron has been shown to be indispensable for the virulence of several fungal pathogens, the siderophore transporter Sit1 was found to mediate uptake of the novel antifungal drug VL-2397, and siderophores were shown to be useful as biomarkers as well as for imaging of fungal infections. However, siderophore uptake in filamentous fungi is poorly characterized. The opportunistic human pathogen Aspergillus fumigatus possesses five putative siderophore transporters. Here, we demonstrate that the siderophore transporters Sit1 and Sit2 have overlapping, as well as unique, substrate specificities. With respect to ferrichrome-type siderophores, the utilization of ferrirhodin and ferrirubin depended exclusively on Sit2, use of ferrichrome A depended mainly on Sit1, and utilization of ferrichrome, ferricrocin, and ferrichrysin was mediated by both transporters. Moreover, both Sit1 and Sit2 mediated use of the coprogen-type siderophores coprogen and coprogen B, while only Sit1 transported the bacterial ferrioxamine-type xenosiderophores ferrioxamines B, G, and E. Neither Sit1 nor Sit2 were important for the utilization of the endogenous siderophores fusarinine C and triacetylfusarinine C. Furthermore, A. fumigatus was found to lack utilization of the xenosiderophores schizokinen, basidiochrome, rhizoferrin, ornibactin, rhodotorulic acid, and enterobactin. Taken together, this study characterized siderophore use by A. fumigatus and substrate characteristics of Sit1 and Sit2.
... Complete medium (2% [wt/ vol] glucose, 0.2% [wt/vol] peptone, 0.1% [wt/vol] yeast extract, 0.1% [wt/vol] Casamino Acids, 7 mM KCl, 2 mM MgSO 4 , 11mM KH 2 PO 4 , and trace elements as described in the recipe for AMM but without iron, with pH adjusted with HCl to 6.5) was used to obtain conidia for RNA isolation and Northern blot analysis. RNA isolation and Northern blot analysis, using 10mg of extracted RNA, were performed essentially as described previously (65). The digoxigenin-labeled hybridization probes used in this study were generated by PCR. ...
Asp f3 is one of the most abundant proteins in the pathogenic mold Aspergillus fumigatus . It has an enigmatic multifaceted role as a fungal allergen, virulence factor, reactive oxygen species (ROS) scavenger, and vaccine candidate.
... Interestingly the oxidative stress induced by CdCl 2 treatment ( Figure 1B) was not accompanied by bulk up-regulation of genes encoding antioxidative enzymes (Table 3 and Table S5). Some genes (e.g., sodB, manganese-superoxide dismutase [53]; ccp1, putative cytochrome c peroxidase [54]; catB and catC, catalases [55]) were up-regulated, while others (e.g., trxA, thioredoxin [56]; gpxA, putative glutathione peroxidase [54]; cpeA, putative catalase-peroxidase [54,55]) were down-regulated (Table S5). These transcriptional changes were accompanied with increased specific SOD activities (Table 1). ...
Cadmium is an exceptionally toxic industrial and environmental pollutant classified as a human carcinogen. In order to provide insight into how we can keep our environment safe from cadmium contamination and prevent the accumulation of it in the food chain, we aim to elucidate how Aspergillus nidulans, one of the most abundant fungi in soil, survives and handles cadmium stress. As AtfA is the main transcription factor governing stress responses in A. nidulans, we examined genome-wide expression responses of wild-type and the atfA null mutant exposed to CdCl2. Both strains showed up-regulation of the crpA Cu2+/Cd2+ pump gene and AN7729 predicted to encode a putative bis(glutathionato)-cadmium transporter, and transcriptional changes associated with elevated intracellular Cys availability leading to the efficient adaptation to Cd2+. Although the deletion of atfA did not alter the cadmium tolerance of the fungus, the cadmium stress response of the mutant differed from that of a reference strain. Promoter and transcriptional analyses of the “Phospho-relay response regulator” genes suggest that the AtfA-dependent regulation of these genes can be relevant in this phenomenon. We concluded that the regulatory network of A. nidulans has a high flexibility allowing the fungus to adapt efficiently to stress both in the presence and absence of this important transcription factor.
... Two main repressors for this purpose have been described: the GATA TF SreA, which downregulates acquisition [65], and the HapX to downregulate iron consumption [66]. During iron sufficiency, SreA represses high-affinity iron uptake and upregulates the consumption of iron via heme biosynthesis, respiration, and ribosome biogenesis [65,76], and HapX upregulates vacuolar storage [77]. During iron starvation, HapX interacts with the DNA-binding CCAAT-binding complex (CBC) to activate RIA, upregulates the synthesis and uptake of siderophores, and represses the iron-consuming pathways [66,77,78]. ...
Aspergillus fumigatus is a ubiquitous soil decomposer and an opportunistic pathogen that is characterized by its large metabolic machinery for acquiring nutrients from media. Lately, an ever-increasing number of genes involved in fungal nutrition has been associated with its virulence. Of these, nitrogen, iron, and zinc metabolism-related genes are particularly noteworthy, since 78% of them have a direct implication in virulence. In this review, we describe the sensing, uptake and regulation process of the acquisition of these nutrients, the connections between pathways and the virulence-implicated genes. Nevertheless, only 40% of the genes mentioned in this review have been assayed for roles in virulence, leaving a wide field of knowledge that remains uncertain and might offer new therapeutic and diagnostic targets.
... GATA TFs AreB and AreA are not only involved in nitrogen and carbon metabolism, but also in the control of several complex cellular processes such as transport and secondary metabolism (SM) (Pfannmüller et al. 2017;Chudzicka-Ormaniec et al. 2019). SreA is involved in the regulation of siderophore biosynthesis and iron uptake (Oberegger et al. 2010;Schrettl et al. 2008), and NsdD regulates sexual and/or asexual reproduction and the production of SMs (Lee et al. 2014(Lee et al. , 2016Niehaus et al. 2017). Furthermore, few fungal GATA TFs also play important roles in response to abiotic stresses. ...
GATA transcription factors (TFs) are involved in the regulation of growth processes and various environmental stresses. Although GATA TFs involved in abiotic stress in plants and some fungi have been analyzed, information regarding GATA TFs in Aspergillus oryzae is extremely poor. In this study, we identified and functionally characterized seven GATA proteins from A. oryzae 3.042 genome, including a novel AoSnf5 GATA TF with 20-residue between the Cys-X 2 -Cys motifs which was found in Aspergillus GATA TFs for the first time. Phylogenetic analysis indicated that these seven A . oryzae GATA TFs could be classified into six subgroups. Analysis of conserved motifs demonstrated that Aspergillus GATA TFs with similar motif compositions clustered in one subgroup, suggesting that they might possess similar genetic functions, further confirming the accuracy of the phylogenetic relationship. Furthermore, the expression patterns of seven A. oryzae GATA TFs under temperature and salt stresses indicated that A. oryzae GATA TFs were mainly responsive to high temperature and high salt stress. The protein–protein interaction network of A. oryzae GATA TFs revealed certain potentially interacting proteins. The comprehensive analysis of A. oryzae GATA TFs will be beneficial for understanding their biological function and evolutionary features and provide an important starting point to further understand the role of GATA TFs in the regulation of distinct environmental conditions in A. oryzae .
... Uniquely, L. corymbifera was shown to possess four putative copies of the iron permease FTR1 [48]. The second mechanism is the ability to produce and/or utilize low molecular weight siderophores (iron chelators) while the third employs heme oxygenase to liberate iron from heme [52][53][54][55][56]. As such, these pathways are known as virulence determinants for the fungal pathogens [57][58][59][60][61]. ...
Iron is an essential micronutrient for most organisms and fungi are no exception. Iron uptake by fungi is facilitated by receptor-mediated internalization of siderophores, heme and reductive iron assimilation (RIA). The RIA employs three protein groups: (i) the ferric reductases (Fre5 proteins), (ii) the multicopper ferroxidases (Fet3) and (iii) the high-affinity iron permeases (Ftr1). Phenotyping under different iron concentrations revealed detrimental effects on spore swelling and hyphal formation under iron depletion, but yeast-like morphology under iron excess. Since access to iron is limited during pathogenesis, pathogens are placed under stress due to nutrient limitations. To combat this, gene duplication and differential gene expression of key iron uptake genes are utilized to acquire iron against the deleterious effects of iron depletion. In the genome of the human pathogenic fungus L. corymbifera, three, four and three copies were identified for FRE5, FTR1 and FET3 genes, respectively. As in other fungi, FET3 and FTR1 are syntenic and co-expressed in L. corymbifera. Expression of FRE5, FTR1 and FET3 genes is highly up-regulated during iron limitation (Fe-), but lower during iron excess (Fe+). Fe- dependent upregulation of gene expression takes place in LcFRE5 II and III, LcFTR1 I and II, as well as LcFET3 I and II suggesting a functional role in pathogenesis. The syntenic LcFTR1 I–LcFET3 I gene pair is co-expressed during germination, whereas LcFTR1 II- LcFET3 II is co-expressed during hyphal proliferation. LcFTR1 I, II and IV were overexpressed in Saccharomyces cerevisiae to represent high and moderate expression of intracellular transport of Fe3+, respectively. Challenge of macrophages with the yeast mutants revealed no obvious role for LcFTR1 I, but possible functions of LcFTR1 II and IVs in recognition by macrophages. RIA expression pattern was used for a new model of interaction between L. corymbifera and macrophages.
... Similar to SRE, iron regulatory roles for SREA were identified by confirming hypersensitivity to the iron-activated antibiotics phleomycin and streptonigrin, and observing accumulation of iron in the mutant lacking the sreA gene (Haas et al. 1999). Moreover, an additional study identified subsets of genes that are dysregulated by deletion of sreA in A. nidulans, and these include genes involved not only in the production and uptake of siderophores, but also production of the intracellular siderophore, ferricrocin, as well as the antioxidative enzymes catalase and superoxide dismutase (Oberegger et al. 2001). These results further confirm the negative regulatory roles of SREA in iron uptake in A. nidulans. ...
Iron acquisition is critical for pathogenic fungi to adapt to and survive within the host environment. However, to same extent, the fungi must also avoid the detrimental effects caused by excess iron. The importance of iron has been demonstrated for the physiology and virulence of major fungal pathogens of humans including Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans. In particular, numerous studies have revealed that aspects of iron acquisition, metabolism, and homeostasis in the fungal pathogens are tightly controlled by conserved transcriptional regulators including a GATA-type iron transcription factor and the CCAAT-binding complex (CBC)/HapX orthologous protein complex. However, the specific downstream regulatory networks are slightly different in each fungus. In addition, roles have been proposed or demonstrated for other factors including monothiol glutaredoxins, BolA-like proteins, and Fe-S cluster incorporation on the GATA-type iron transcription factor and the CBC/HapX orthologous protein complex, although limited information is available. Here we focus on recent work on C. neoformans in the context of an emerging framework for fungal regulation of iron acquisition, metabolism, and homeostasis. Our specific goal is to summarize recent findings on transcriptional networks governed by the iron regulators Cir1 and HapX in C. neoformans.
... Also studies on genes and gene mutation clearly depicted the contrasting functions of intracellular and extracellular siderophores in maintaining the iron homeostasis. Furthermore, the cellular energy uptake for the siderophore biosynthesis is remarkably high in microbes; hence to sustain the situation, a tight regulation might be followed under significant iron shortage (Oberegger et al. 2001). ...
Iron homeostasis is an important process in many living organisms. Siderophores are the secondary metabolites produced by fungi essential to access the cellular components of the host for growth and development. Siderophores are encoded by different genes in the organism and perform different functions like extracellular iron acquisition, intracellular iron storage, conidial iron acquisition, as well as storage during infections. It has been shown that mutations in siderophore-coding gene have definite effect on fungal growth and viability. In recent era, fungal siderophores are highly focused area of research owing to its wide applicability in the field of agricultural sectors, environmental impacts, and health-care sectors. However, because of the limitation in culturing the fungal strains in laboratories, only about 5% of the fungal diversity has been partially exploited. Fungal research would offer an enormous source of novelty if the constraints of their isolation and culturing could be overcome. The potential in the siderophore research is high as there is a lacuna in the understanding of the siderophoric applications in various fields. This chapter aims to highlight the importance of siderophore research and their potential application to be considered for further work.
... GATA TFs AreB and AreA are not only involved in the nitrogen and carbon metabolism, but also in the control of several complex cellular processes such as transport and secondary metabolism (SM) (Pfannmüller et al. 2017; Chudzicka-Ormaniec et al. 2019). The SreA involves in regulation of siderophore biosynthesis and iron uptake (Oberegger et al. 2010;Schrettl et al. 2008), and NsdD regulates sexual and/or asexual reproduction and the production of SMs (Lee et al. 2014;Niehaus et al. 2017). Furthermore, few fungal GATA TFs also play important role in response to the abiotic stresses. ...
GATA transcription factors（TFs）are involved in the regulation of diverse growth processes and various environmental stimuli stresses. Although the analysis of GATA TFs involved in abiotic stress has been performed in plants and some fungi, information regarding GATA TFs in A. oryzae is extremely poor. Therefore, we identified seven GATA TFs from A. oryzae 3.042 genome and classified into six subgroups in NJ_tree, including a novel AoSnf5 with 20-residue between the Cys-X 2 -Cys motifs which was found in Aspergillus for the first time. Conserved motifs demonstrated that Aspergillus GATA TFs with similar motif compositions clustered into one subgroup, which suggests they might have similar genetic functions and further confirms the accuracy of the phylogenetic relationship. Moreover, the expression patterns of seven A. oryzae GATA TFs under temperature and salt stresses indicated that A. oryzae GATA TFs were mainly responsive to high-temperature and high salt stress. The PPI network of A. oryzae GATA TFs proposed some potentially interacting proteins. The comprehensive analysis of A. oryzae GATA TFs will be beneficial to understand their functional and evolutionary features and provide useful information for the further analyzing the role of GATA TFs in regulation of distinct environmental conditions in A. oryzae .
... Research has demonstrated that the AreB and AreA GATA TFs are regulators that are not only involved in the nitrogen and carbon metabolism, but also in the control of several complex cellular processes such as transport and secondary metabolism in lamentous fungi [11,12]. The SreA involves in regulation of siderophore biosynthesis and the control of iron uptake [10,14], and NsdD is a global regulator that regulates sexual and/or asexual reproduction and the production of SMs in A. nidulans and A. fumigatus [15,16]. Fungal GATA TFs are a combination of both plant and animal GATA TFs in terms of amino acid residues present in the zinc-nger loop [5,6,8]. ...
Background
GATA transcription factors (TFs) are transcriptional regulatory proteins that contain a characteristic type-IV zinc finger and recognize the conserved GATA motif in the promoter region. Previous studies demonstrate that GATA TFs are involved in the regulation of diverse growth processes and various environmental stimuli stresses. Although the analysis of GATA TFs involved in abiotic stress have been performed in model plants and some fungi, information regarding GATA TFs in A. oryzae is extremely poor.
Results
Therefore, we identified seven GATA TFs from A. oryzae 3.042 genome, and named AoAreA, AoAreB, AoLreA, AoLreB, AoNsdD, AoSreA in correspondence to fungal orthologs, including a novel AoSnf5 with 20-residue between the Cys-X2-Cys motifs which was found in Aspergillus for the first time. Six known A. oryzae GATA TFs were classified into six subgroups, while the novel AoSnf5 also clustered into NSDD subgroups together with AoNsdD in the NJ_tree of all Aspergillus GATA TFs. Conserved motifs demonstrated that GATA TFs with similar motif compositions clustered into one subgroup, which suggests they might have similar genetic functions and further confirms the accuracy of the phylogenetic relationship of Aspergillus GATA TFs. The expression patterns of seven A. oryzae GATA TFs exhibited diversity under temperature and salt stresses. The expression analyses of AoLreA and AoLreB demonstrates AoLreA mainly played role in salt stress and AoLreB did under temperature stress. AoSreA was shown to positively regulate the expression of AoCreA and might act as a negative regulator in temperature and high salt stress response. In addition, the AoNsdD, AoSnf5, AoAreB, and AoAreA strongly responsed to salt stresses, while AoAreB and AoAreA showed opposite expression trends at high temperature. Overall, the expression patterns of these A. oryzae GATA TFs under distinct environmental conditions provided useful information for the further analysis of GATA TFs in regulation of various abiotic stress in A. oryzae.
Conclusion
In conclusion, the comprehensive analysis data of A. oryzae GATA TFs will provide insights into the critical role of A. oryzae GATA TFs in resistance to temperature and salt stresses in A. oryzae.
... RNA was isolated using TRI Reagent (Sigma-Aldrich, Vienna, Austria) and peqGOLD Phase Trap (VWR Peqlab, Vienna, Austria) reaction tubes. Ten micrograms of total RNA were analyzed as described previously [34]. Digoxigenin-labeled hybridization probes were amplified by PCR using primers listed in Table S2. ...
Aspergillus fumigatus is an opportunistic human pathogen mainly infecting immunocompromised patients. The aim of this study was to characterize the role of arginine biosynthesis in virulence of A. fumigatus via genetic inactivation of two key arginine biosynthetic enzymes, the bifunctional acetylglutamate synthase/ornithine acetyltransferase (argJ/AFUA_5G08120) and the ornithine carbamoyltransferase (argB/AFUA_4G07190). Arginine biosynthesis is intimately linked to the biosynthesis of ornithine, a precursor for siderophore production that has previously been shown to be essential for virulence in A. fumigatus. ArgJ is of particular interest as it is the only arginine biosynthetic enzyme lacking mammalian homologs. Inactivation of either ArgJ or ArgB resulted in arginine auxotrophy. Lack of ArgJ, which is essential for mitochondrial ornithine biosynthesis, significantly decreased siderophore production during limited arginine supply with glutamine as nitrogen source, but not with arginine as sole nitrogen source. In contrast, siderophore production reached wild-type levels under both growth conditions in ArgB null strains. These data indicate that siderophore biosynthesis is mainly fueled by mitochondrial ornithine production during limited arginine availability, but by cytosolic ornithine production during high arginine availability via cytosolic arginine hydrolysis. Lack of ArgJ or ArgB attenuated virulence of A. fumigatus in the insect model Galleria mellonella and in murine models for invasive aspergillosis, indicating limited arginine availability in the investigated host niches.
Plant pathogens are challenged by host‐derived iron starvation or excess during infection, but the mechanism of plant pathogens rapidly adapting to the dynamic host iron environments to assimilate iron for invasion and colonization remains largely unexplored. Here, we found that the GATA transcription factor SreC in Curvularia lunata is required for virulence and adaption to the host iron excess environment. SreC directly binds to the ATGWGATAW element in an iron‐dependent manner to regulate the switch between different iron assimilation pathways, conferring adaption to host iron environments in different trophic stages of C. lunata . SreC also regulates the transition of trophic stages and developmental processes in C. lunata . SreC‐dependent adaption to host iron environments is essential to the infectious growth and survival of C. lunata . We also demonstrate that CgSreA (a SreC orthologue) plays a similar role in Colletotrichum graminicola . We conclude that Sre mediates adaption to the host iron environment during infection, and the function is conserved in hemibiotrophic fungi.
Fungi can be found in virtually every habitat on earth. Over time, they evolved strategies to survive even the most challenging conditions, leading to their enormous diversity. For their survival in the habitat they have acquired the ability to produce a multitude of secondary metabolites, also named natural products. These unusual low-molecular-weight compounds exhibit a variety of effects, ranging from antimicrobial activity to protective compounds and information molecules for neighboring microorganisms. Through these effects, our recent data substantiate the hypothesis that secondary metabolites shape the composition of microbial consortia (microbiomes) by reducing or promoting the growth of certain microorganisms or changing their metabolic activity. Genetic analyses indicate that fungi have the potential to produce far more secondary metabolites than have been identified yet. Their encoding biosynthesis gene clusters remain silent under laboratory conditions and the ecological context is required for their activation. Therefore, we have initiated research on the activation of such silent gene clusters by microbial communication. Since then, many studies attempted to mimic naturally inducing conditions, e.g., by varying culture conditions, genetic manipulation of the producing organism, or co-culturing of multiple microorganisms. Elucidating the ecological roles of these compounds and the underlying triggers leading to their production is of major importance for our understanding of how microbial communities are shaped and how complex interactive networks can be formed with their profound influence on human health and the environment. Further, understanding the ecological trigger regulating the production of secondary metabolites will allow us to shed light on the pool of yet unidentified compounds in search for potential new antibiotics and other useful compounds.KeywordsSecondary metabolitesFungiSilent BGCsMicrobial interactionCross-kingdom communicationMicrobiomeChemical signalingInfectionMycotoxinsAntibiotics
Iron is one of the essential nutrients for almost all microorganisms. Under iron-limited conditions, bacteria can secrete siderophores to the outside world to absorb iron for survival. This process requires the coordinated action of energy-transducing proteins, transporters, and receptors. The spoilage factors of some spoilage bacteria and the pathogenic mechanism of pathogenic bacteria are also closely related to siderophores. Meanwhile, some siderophores have also gradually evolved toward beneficial aspects. First, a variety of siderophores are classified into three aspects. In addition, representative iron uptake systems of Gram-negative and Gram-positive bacteria are described in detail to understand the common and specific pathways of iron uptake by various bacteria. In particular, the causes of siderophore-induced bacterial pathogenicity and the methods and mechanisms of inhibiting bacterial iron absorption under the involvement of siderophores are presented. Then, the application of siderophores in the food sector is mainly discussed, such as improving the food quality of dairy products and meat, inhibiting the attack of pathogenic bacteria on food, improving the plant growth environment, and promoting plant growth. Finally, this review highlights the unresolved fate of siderophores in the iron uptake system and emphasizes further development of siderophore-based substitutes for traditional drugs, new antibiotic-resistance drugs, and vaccines in the food and health sectors.
Many living organisms have important iron homeostasis processes. The secondary metabolites known as siderophores are created by fungi and are crucial for gaining access to the host’s cellular components for growth and development. The organism’s siderophores are proteins that have multiple roles, including acquiring external iron, storing it intracellularly, acquiring it via conidia, and storing it during infections. It has been demonstrated that changes in the siderophore-coding gene have a real impact on the health and viability of the fungus. Due to their extensive application in the agricultural, environmental, and medical sectors, fungal siderophores are currently a highly concentrated field of research. However, only around 5% of the fungal diversity has been partially utilized due to the difficulties in growing the fungal strains in lab settings. If their isolation and culturing limitations could be removed, fungal research would provide a huge source of novelty. Due to a knowledge gap regarding siderophore applications in various fields, siderophore research has great potential.KeywordsSiderophoresFungiPlant healthPlant immunityIron
Antrodia cinnamomea is a precious edible and medicinal fungus with activities of antitumor, antivirus, and immunoregulation. Fe2+ was found to promote the asexual sporulation of A. cinnamomea markedly, but the molecular regulatory mechanism of the effect is unclear. In the present study, comparative transcriptomics analysis using RNA sequencing (RNA-seq) and real time quantitative PCR (RT-qPCR) were conducted on A. cinnamomea mycelia cultured in the presence or absence of Fe2+ to reveal the molecular regulatory mechanisms underlying iron-ion-promoted asexual sporulation. The obtained mechanism is as follows: A. cinnamomea acquires iron ions through reductive iron assimilation (RIA) and siderophore-mediated iron assimilation (SIA). In RIA, ferrous iron ions are directly transported into cells by the high-affinity protein complex formed by a ferroxidase (FetC) and an Fe transporter permease (FtrA). In SIA, siderophores are secreted externally to chelate the iron in the extracellular environment. Then, the chelates are transported into cells through the siderophore channels (Sit1/MirB) on the cell membrane and hydrolyzed by a hydrolase (EstB) in the cell to release iron ions. The O-methyltransferase TpcA and the regulatory protein URBS1 promote the synthesis of siderophores. HapX and SreA respond to and maintain the balance of the intercellular concentration of iron ions. Furthermore, HapX and SreA promote the expression of flbD and abaA, respectively. In addition, iron ions promote the expression of relevant genes in the cell wall integrity signaling pathway, thereby accelerating the cell wall synthesis and maturation of spores. This study contributes to the rational adjustment and control of the sporulation of A. cinnamomea and thereby improves the efficiency of the preparation of inoculum for submerged fermentation.
Endophytic fungal communities have attracted a great attention to chemists, ecologists, and microbiologists as a treasure trove of biological resource. Endophytic fungi play incredible roles in the ecosystem including abiotic and biotic stress tolerance, eco-adaptation, enhancing growth and development, and maintaining the health of their host. In recent times, endophytic fungi have drawn a special focus owing to their indispensable diversity, unique distribution, and unparalleled metabolic pathways. The endophytic fungal communities belong to three phyla, namely Mucoromycota, Basidiomycota, and Ascomycota with seven predominant classes Agaricomycetes, Dothideomycetes, Eurotiomycetes, Mortierellomycotina, Mucoromycotina, Saccharomycetes, and Sordariomycetes. In a review of a huge number of research finding, it was found that endophytic fungal communities of genera Aspergillus, Chaetomium, Fusarium, Gaeumannomyces, Metarhizium, Microsphaeropsis, Paecilomyces, Penicillium, Piriformospora, Talaromyces, Trichoderma, Verticillium, and Xylaria have been sorted out and well characterized for diverse biotechnological applications for future development. Furthermore, these communities are remarkable source of novel bioactive compounds with amazing biological activity for use in agriculture, food, and pharmaceutical industry. Endophytes are endowed with a broad range of structurally unique bioactive natural products, including alkaloids, benzopyranones, chinones, flavonoids, phenolic acids, and quinines. Subsequently, there is still an excellent opportunity to explore novel compounds from endophytic fungi among numerous plants inhabiting different niches. Furthermore, high-throughput sequencing could be a tool to study interaction between plants and endophytic fungi which may provide further opportunities to reveal unknown functions of endophytic fungal communities. The present review deals with the biodiversity of endophytic fungal communities and their biotechnological implications for agro-environmental sustainability.
Fungi are universally present; however, till date only few species have been explored for human welfare, e.g., penicillin, a secondary metabolite that has had a great impact on human civilization. It was a groundbreaking discovery that enabled the researches to further explore the great biodiversity of fungi and eventually use them for human welfare. Fungi are capable to produce a variety of primary and secondary biomolecules that have great value in medicine, industries, and agriculture. In addition, it has tremendous application in soil and waste water remediation. Fungi produce antibiotics (e.g., penicillin, glycopeptides, quinolones, etc.) and industrially important enzymes (e.g., cellulase, xylanase, protease, etc.), and apart from that, they produce toxins (e.g., ergot, patulin, etc.) which in prescribed amount are used as a lifesaver. In addition to that, the endophytes are also known to produce a huge diversity of metabolites. Thus, the present chapter discusses in detail about the various applications of fungi, and its metabolites such as enzymes and mycotoxins along with its limitation, and future prospect.
In the past few decades, it has been realized through research that fungal siderophores epitomize the uptake of iron as well as other essential elements like zinc, magnesium, copper, nickel and arsenic. Understanding the chemical structures of different fungal siderophores and the membrane receptors involved in uptake of mineral ions has opened new areas for research. In this edited volume, recent research is presented on fungal siderophores in one comprehensive volume to provide researchers a strong base for future research.
Siderophores are the low molecular weight, high affinity iron-chelating compounds produced by bacteria and fungi. They are responsible for transporting iron across the cell membrane. Fungi produce a range of hydroxamate siderophores involved in the uptake of essential elements in almost all microorganisms and plants. In recent years, siderophores have been used in molecular imaging applications to visualize and understand cellular functions, which thus provide an opportunity to identify new drug targets. Therefore, knowledge of fungal siderophores has become vital in current research.
Siderophores have received much attention in recent years because of their potential roles and applications in various research areas. Their significance in these applications is because siderophores have the ability to bind a variety of metals in addition to iron, and they have a wide range of chemical structures and specific properties. For instance, siderophores function as biocontrols, biosensors, and bioremediation and chelation agents, in addition to their important role in weathering soil minerals and enhancing plant growth.
This book focuses on siderophores with the following significant points. It discusses leading, state-of-the-art research in all possible areas on fungal siderophores. The contributors are well-known and recognized authorities in the field of fungal siderophores. It discusses a projection of practical applications of fungal siderophores in various domains. This is the first book exclusively on fungal siderophores.
In this comprehensive, edited volume, we show leading research on fungal siderophores and provide the most recent knowledge of researchers' work on siderophores. This book presents in-depth knowledge on siderophores to researchers working in areas of health sciences, microbiology, plant sciences, biotechnology, and bioinformatics.
Several fungal infections occur in human body and cause various health issues and also create critical impacts to our health. Among these fungal infections, Aspergillus infections are emerging as life-threatening yet underappreciated and underdeveloped as compared to other microbial infections. Aspergillosis is a serious threat to human life and requires more attention in terms of diagnosis and treatment. Siderophores play an important role in fungal virulence and iron acquisition mechanisms and also helps in the process of treatment and therapy of such fungal infections. In this chapter, we discussed various infections caused by Aspergillus species and their virulence in different stages of the disease. The two major Aspergillus pathogens A. fumigatus and A. nidulans were taken for the study, and their siderophoric system involved in biosynthetic pathway and the role of siderophores involved in Aspergillus species has been discussed. From this review, we suggest that fungal siderophore biosynthetic pathway might represent promising therapeutic targets for the treatment of aspergillosis.
Iron (Fe) is an essential nutrient for life and the fourth most abundant element in the earth. The availability of ferric iron (Fe III) is less in soil solution due to the low solubility of ferric hydroxides, oxides, and oxyhydroxides. Therefore, the Fe availability to microbes and plants is limited, albeit its abundance in the environment. Therefore, the availability of Fe to microbes and plants has evolved strategies based on acidification through proton extrusion and organic acid production, chelation, ligands like siderophore and phytosiderophore production, and enzymatic reduction involving reductase enzymes. This review attempts to explain the fungal siderophore, its biosynthesis, transport, and practical application. Thus, siderophore is an iron-binding molecule synthesized by fungi, bacteria, cyanobacteria, and plants. The common types of siderophore are hydroxamates, catecholates, carboxylates, but hydroxamate type is dominant in fungi. L-ornithine is a biosynthetic precursor of siderophore and synthesized through multimodular large enzymes complex nonribosomal peptide synthetase (NRPSs) dependent/independent. Siderophore-Fe chelators protein (FIT1, FIT2, and FIT3) helps in the retention of siderophore. Saccharomyces cerevisiae expresses two genetically separate systems (reductive and a non-reductive system) at the plasma membrane, which converts Fe III into Fe II by ferrous-specific metallo-reductases enzyme complex, FRE reductases. Regulation of the siderophore gene expression on the promoter region by Fur Box protein depends on the availability of Fe in the external medium. Biotechnologically, it is more important due to its wide range of applications that include medical, remediation of heavy metal, biocontrol of plant pathogens, and enzyme inhibitions.
As the most toxic and carcinogenic mycotoxin, aflatoxin B1 (AFB1) biosynthesis depends on a series of enzymatic reactions and a complicated regulatory system. Methyl jasmonate (MeJA) is one of stress associated phytohormones. In this study, MeJA could inhibit A. flavus growth and AFB1 production with a dose-dependent manner. SEM and TEM analysis indicated that morphological ultrastructure deteriorations were observed in A. flavus treated with MeJA. RNA-Seq indicated that the initial-steps aflatoxins (AFs) genes were no drastic difference, but the middle- and later- steps genes were significantly down-regulated, which might be due to the decreases of global regulators, especially AtfB. More importantly, two novel regulators (AFLA_085880 and AFLA_015850) were involved in the inhibition, and were recognized as the critically positive regulators for AFs productions. The two genes mutants also showed significantly decrease expressions of AFs cluster genes and AFs associated regulators, and subsequent AFB1 biosynthesis. This research partly clarified inhibitory mechanism of MeJA and made some contributions to the elimination of AFs contamination.
To maintain iron homeostasis, fungi have to balance iron acquisition, storage, and utilization to ensure sufficient supply and to avoid toxic excess of this essential trace element. As pathogens usually encounter iron limitation in the host niche, this metal plays a particular role during virulence. Siderophores are iron-chelators synthesized by most, but not all fungal species to sequester iron extra- and intracellularly. In recent years, the facultative human pathogen Aspergillus fumigatus has become a model for fungal iron homeostasis of siderophore-producing fungal species. This article summarizes the knowledge on fungal iron homeostasis and its links to virulence with a focus on A. fumigatus. It covers mechanisms for iron acquisition, storage, and detoxification, as well as the modes of transcriptional iron regulation and iron sensing in A. fumigatus in comparison to other fungal species. Moreover, potential translational applications of the peculiarities of fungal iron metabolism for treatment and diagnosis of fungal infections is addressed.
Background
Fungal GATA-type transcription factors (GATA-TFs) are a class of transcriptional regulators involved in various biological processes. However, their functions are rarely analyzed systematically, especially in edible or medicinal fungi, such as Tolypocladium guangdongense , which has various medicinal and food safety properties with a broad range of potential applications in healthcare products and the pharmaceutical industry.
Methods
GATA-TFs in T. guangdongense (TgGATAs) were identified using InterProScan. The type, distribution, and gene structure of TgGATAs were analyzed by genome-wide analyses. A phylogenetic tree was constructed to analyze their evolutionary relationships using the neighbor-joining (NJ) method. To explore the functions of GATA-TFs, conserved domains were analyzed using MEME, and cis-elements were predicted using the PlantCARE database. In addition, the expression patterns of TgGATAs under different light conditions and developmental stages were studied using qPCR.
Results
Seven TgGATAs were identified. They were randomly distributed on four chromosomes and contained one to four exons. Phylogenetic analysis indicated that GATA-TFs in each subgroup are highly conserved, especially for GATA1 to GATA5. Intron distribution analyses suggested that GATA1 and GATA3 possessed the most conserved gene structures. Light treatments induced the expression levels of TgGATA1 and TgGATA5-7 , but the expression levels varied depending on the duration of illumination. The predicted protein structures indicate that TgGATA1 and TgGATA2 possess typical light-responsive domains and may function as photoreceptors to regulate downstream biological processes. TgGATA3 and TgGATA5 may be involved in nitrogen metabolism and siderophore biosynthesis, respectively. TgGATA6 and TgGATA7 possess unique Zn finger loop sequences, suggesting that they may have special functions. Furthermore, gene expression analysis indicated that TgGATA1 ( WC1 ) was notably involved in mycelial color transformation, while other genes were involved in fruiting body development to some extent. These results provide valuable information to further explore the mechanisms through which TgGATAs are regulated during fruiting body development.
Marker genes are essential for gene modification and genome editing of microorganisms. In Aspergillus oryzae, a widely used host for enzyme production, only a few marker genes can be used for positive selection. One of these genes, the pyrithiamine (PT) resistance marker gene thiA, is not useful for CRISPR/Cas9 genome editing because of its unique resistance-conferring mechanism. In this study, a novel PT resistance marker was investigated considering its potential applications in genome editing. A mutant resistant to PT was selected from UV-mutagenized A. oryzae RIB40. Whole genome analysis was conducted on the mutants, and a novel candidate gene for PT resistance was identified. This candidate gene exhibited similarity to the thiamine transporter gene thi9 of Schizosaccharomyces pombe and was designated as thiI. A thiI loss-of-function mutant was generated using the CRISPR/Cas9 genome editing system to investigate its effect on PT resistance. This mutant showed PT resistance and exhibited no growth defect or auxotrophy. The thiI gene was further investigated for its use as a selection marker in genome co-editing. Ribonucleoprotein complex comprising recombinant Cas9 nuclease and sgRNA targeting thiI or another target gene (wA or sreA) was prepared and simultaneously introduced into A. oryzae RIB40. thiI and target gene double loss-of-function mutants were efficiently selected on PT-containing medium. thiI was shown to be a useful marker gene in A. oryzae for use in genome editing. This study is expected to provide insights, which will promote basic research and industrial applications of A. oryzae.
Neurospora crassa produces several structurally distinct siderophores: coprogen, ferricrocin, ferrichrome C and some minor unknown compounds. Under conditions of iron starvation, desferricoprogen is the major extracellular siderophore whereas desferriferricrocin and desferriferrichrome C are predominantly found intracellularly. Mssbauer spectroscopic analyses revealed that coprogen-bound iron is rapidly released after uptake in mycelia of the wild-typeN.crassa 74A. The major intracellular target of iron distribution is desferriferricrocin. No ferritin-like iron pools could be detected. Ferricrocin functions as the main intracellular iron-storage peptide in mycelia ofN. crassa. After uptake of ferricrocin in both the wild-typeN. crassa 74A and the siderophore-free mutantN. crassa arg-5 ota aga, surprisingly little metabolization (11%) could be observed. Since ferricrocin is the main iron-storage compound in spores ofN. crassa, we suggest that ferricrocin is stored in mycelia for inclusion into conidiospores.
Transport proteins of microorganisms may either belong to the ATP-binding cassette (ABC) superfamily or to the major facilitator (MFS)-superfamily. MFS transporters are single-polypeptide membrane transporters that transport small molecules via uniport, symport or antiport mechanisms in response to a chemiosmotic gradient. Although Saccharomyces cerevisiae is a non-siderophore producer, various bacterial and fungal siderophores can be utilized as an iron source. From yeast genome sequencing data six genes of the unknown major facilitator (UMF) family were known of which YEL065w Sce was recently identified as a transporter for the bacterial siderophore ferrioxamine B (Sit1p). The present investigation shows that another UMF gene, YHL047c Sce, encodes a transporter for the fungal siderophore triacetylfusarinine C. The gene YHL047c Sce (designated TAF1) was disrupted using the kanMX disruption module in a fet3 background (strain DEY 1394 fet3), possessing a defect in the high affinity ferrous iron transport. Growth promotion assays and transport experiments with 55Fe-labelled triacetylfusarinine C showed a complete loss of iron utilization and uptake in the disrupted strain, indicating that TAF1 is the gene for the fungal triacetylfusarinine transport in Saccharomyces cerevisiae and possibly in other siderophore producing fungi.
In this study, we monitored and compared the uptake of iron in the fungus Ustilago maydis by using biomimetic siderophore analogs of ferrichrome, the fungal native siderophore, and ferrioxamine B (FOB), a xenosiderophore. Ferrichrome-iron was taken up at a higher rate than FOB-iron. Unlike ferrichrome-mediated uptake, FOB-mediated iron transport involved an extracellular reduction mechanism. By using fluorescently labeled siderophore analogs, we monitored the time course, as well as the localization, of iron uptake processes within the fungal cells. A fluorescently labeled ferrichrome analog, B9-lissamine rhodamine B, which does not exhibit fluorescence quenching upon iron binding, was used to monitor the entry of the compounds into the fungal cells. The fluorescence was found intracellularly 4 h after the application and later was found concentrated in two to three vesicles within each cell. The fluorescence of the fluorescently labeled FOB analog CAT18, which is quenched by iron, was visualized around the cell membrane after 4 h of incubation with the ferrated (nonfluorescent) compounds. This fluorescence intensity increased with time, demonstrating fungal iron uptake from the siderophores, which remained extracellular. We here introduce the use of fluorescent biomimetic siderophores as tools to directly track and discriminate between different pathways of iron uptake in cells.
Three strains of the fungus Aspergillus, Aspergillus quadricinctus (E. Yuill), A. fumigatus (Fresenius), and A. melleus (Yukawa), each producing different iron-chelating compounds during iron-deficient cultivation, were used for 55Fe3+ uptake measurements. Iron from chelates of the ferrichrome-type family was taken up by young mycelia of all strains tested, irrespective of the ferrichrome-type compound these strains predominantly produce in low-iron cultures. Ferrichrysin-producing strains, however, seem to favor ferrichrysin iron uptake, whereas ferrichrome, ferricrocin, and even ferrirubin showed similar iron transport properties in all of these strains. Compared to iron uptake from ferrichrome-type compounds (Km approximately 4 uM) iron uptake from fusigen revealed completely different kinetic values (Km approximately 50 to 80 muM). Iron from exogenous chelates, e.g., from coprogen produced by Neurospora crassa for ferrioxamine B produced by Streptomyces pilosus, can obviously not be taken up by Aspergillus, confirming the pronounced specificity of chelate-iron transport in fungi.
The Escherichia coli ferric enterobactin esterase gene (fes) was cloned into the vector pGEM3Z under the control of the T7 gene 10 promoter and overexpressed to approximately 15% of the total cellular protein. The ferric enterobactin esterase (Fes) enzyme was purified as a 43-kDa monomer by gel filtration chromatography. Purified Fes preparations were examined for esterase activity on enterobactin and its metal complexes and for iron reduction from ferric complexes of enterobactin and 1,3,5-tris(N,N',N"-2,3-dihydroxybenzoyl)aminomethylbenzene (MECAM), a structural analog lacking ester linkages. Fes effectively catalyzed the hydrolysis of both enterobactin and its ferric complex, exhibiting a 4-fold greater activity on the free ligand. It also cleaved the aluminum (III) complex at a rate similar to the ferric complex, suggesting that ester hydrolysis of the ligand backbone is independent of any reductive process associated with the bound metal. Ferrous iron was released from the enterobactin complex at a rate similar to ligand cleavage indicating that hydrolysis and iron reduction are tightly associated. However, no detectable release of ferrous iron from the MECAM complex implies that, with these in vitro preparations, metal reduction depends upon, and is subsequent to, the esterase activity of Fes. These observations are discussed in relation to studies which show that such enterobactin analogs can supply growth-promoting iron concentrations to E. coli.
Development of physical genomic maps is facilitated by identification of overlapping recombinant DNA clones containing long
chromosomal DNA inserts. To simplify the analysis required to determine which clones in a genomic library overlap one another,
we partitioned Aspergillus nidulans cosmid libraries into chromosome-specific subcollections. The eight A. nidulans chromosomes were resolved by pulsed field gel electrophoresis and hybridized to filter replicas of cosmid libraries. The
subcollections obtained appeared to be representative of the chromosomes based on the correspondence between subcollection
size and chromosome length. A sufficient number of clones was obtained in each chromosome-specific subcollection to predict
the overlap and assembly of individual clones into a limited number of contiguous regions. This approach should be applicable
to many organisms whose genomes can be resolved by pulsed field gel electrophoresis.
Control of expression of the Saccharomyces cerevisiae CTT1 (catalase T) gene by the HAP1 (CYP1) gene, a mediator of heme control of mitochondrial cytochromes, was studied. Expression of a CTT1-lacZ fusion in a hap1 mutant showed that the CTT1 promoter is under HAP1 control. As demonstrated by a gel retardation assay, the HAP1 protein binds to a heme control region of the CTT1 gene. This binding in vitro is stimulated by hemin. The HAP1-binding sequence was localized by using DNA fragments spanning different regions, by DNase I footprinting and by methylation interference of DNA-protein binding. The binding site was compared to the HAP1-binding sequences previously characterized in detail (UAS1CYC1, UASCYC7). There is strikingly little similarity between the three sequences, which have only four of those 23 bp in common which are protected from DNase I digestion. However, the pattern of major and minor groove contacts in the complex is quite similar in all three cases. The results obtained show that there is true co-ordinate control of expression of mitochondrial cytochromes and at least some extra-mitochondrial hemoproteins. Heme acts as a metabolic signal in this coordination, which is mediated by the HAP1 protein.
Spores of Neurospora crassa 74A are lacking in ferritinlike iron pools, as demonstrated by Mössbauer spectroscopic analysis. The cyclic hexapeptide siderophore ferricrocin constituted 47% of the total iron content in spores. After germination and growth, the ferricrocin iron pool disappeared, indicating that the metal was utilized. In spores of Aspergillus ochraceus, 74% of the total iron content was bound by ferrichrome-type siderophores. Siderophores may function as iron storage forms in fungal systems.
A reversed-phase HPLC separation of iron(III) chelates of 16 representative fungal siderophores including ferrichromes, coprogens and triacetylfusarinine C was established in order to investigate siderophore production of fungi. For comparison purposes, the widely used bacterial siderophore ferrioxamine B was included. Culture filtrates of the fungi Penicillium resticulosum, Fusarium dimerum, Aspergillus fumigatus and Neurospora crassa were quantitatively analyzed for the presence of known and unknown siderophores after growth in low-iron culture media and adsorption on XAD-2 columns using this HPLC separation system. Photodiode array detection allowed the distinction between siderophores and non-siderophores. According to their ultraviolet/visible spectra, a further classification of the siderophores into four types due to the number of anhydromevalonic acid residues per molecule (0-3) was possible.
Neurospora crassa produces several structurally distinct siderophores: coprogen, ferricrocin, ferrichrome C and some minor unknown compounds. Under conditions of iron starvation, desferricoprogen is the major extracellular siderophore whereas desferriferricrocin and desferriferichrome C are predominantly found intracellularly. Mössbauer spectroscopic analyses revealed that coprogen-bound iron is rapidly released after uptake in mycelia of the wild-type N. crassa 74A. The major intracellular target of iron distribution is desferriferricrocin. No ferritin-like iron pools could be detected. Ferricrocin functions as the main intracellular iron-storage peptide in mycelia of N. crassa. After uptake of ferricrocin in both the wild-type N. crassa 74A and the siderophore-free mutant N. crassa arg-5 ota aga, surprisingly little metabolization (11%) could be observed. Since ferricrocin is the main iron-storage compound in spores of N. crassa, we suggest that ferricrocin is stored in mycelia for inclusion into conidiospores.
The cyclic trihydroxamic acid, N,N',N''-triacetylfusarinine C, produced by Mycelia sterilia EP-76, was shown to be a ferric ionophore for this organism. The logarithm of the association constant k for the ferric triacetylfusarinine C chelate was determined to be 31.8. Other iron-chelating agents, such as rhodotorulic acid, citric acid, and the monomeric subunit of triacetylfusarinine C, N-acetylfusarinine, delivered iron to the cells by an indirect mechanism involving iron exchange into triacetylfusarinine C. In vitro ferric ion exchange was found to be rapid with triacetylfusarinine C. Gallium uptake rates comparable to those of iron were observed with the chelating agents that transport iron into the cell. Ferrichrome, but not ferrichrome A, was also capable of delivering iron and gallium to this organism, but not by an exchange mechanism. Unlike triacetylfusarinine C, the 14C-ligand of ferrichrome was retained by the cell. A midpoint potential of -690 mV with respect to the saturated silver chloride electrode was obtained for the ferric triacetylfusarinine C complex, indicating that an unfavorable reduction potential was not the reason for the use of a hydrolytic mechanism of intracellular iron release from the ferric triacetylfusarinine C chelate.
Siderophores are common products of aerobic and facultative anaerobic bacteria and of fungi. Elucidation of the molecular genetics of siderophore synthesis, and the regulation of this process by iron, has been facilitated by the fact that E. coli uses its own siderophores as well as those derived from other species, including fungi. Overproduction of the siderophore and its transport system at low iron is in this species well established to be the result of negative transcriptional repression, but the detailed mechanism may be positive in other organisms. Siderophores are transported across the double membrane envelope of E. coli via a gating mechanism linking the inner and outer membranes.
This report describes a new method for simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. The method is based on the use of a reagent containing phenol and guanidine thiocyanate. A biological sample is homogenized in the reagent and the simultaneous isolation of RNA, DNA and proteins is accomplished in a single step by a liquid-phase separation. The isolation of RNA can be completed in about 1 h, and DNA and proteins in about 3 h. The simultaneously isolated RNA, DNA and proteins are ready for Northern, Southern and Western blotting. The complete recovery of DNA from samples used for the RNA and protein isolation makes it possible to normalize the results of gene expression studies based on DNA content instead of on the more variable total RNA, protein content or tissue weight.
The Escherichia coli Fur protein, with its iron(II) cofactor, represses iron assimilation and manganese superoxide dismutase (MnSOD) genes, thus coupling iron metabolism to protection against oxygen toxicity. Iron assimilation is triggered by iron starvation in wild-type cells and is constitutive in fur mutants. We show that iron metabolism deregulation in fur mutants produces an iron overload, leading to oxidative stress and DNA damage including lethal and mutagenic lesions. fur recA mutants were not viable under aerobic conditions and died after a shift from anaerobiosis to aerobiosis. Reduction of the intracellular iron concentration by an iron chelator (ferrozine), by inhibition of ferric iron transport (tonB mutants), or by overexpression of the iron storage ferritin H-like (FTN) protein eliminated oxygen sensitivity. Hydroxyl radical scavengers dimethyl sulfoxide and thiourea also provided protection. Functional recombinational repair was necessary for protection, but SOS induction was not involved. Oxygen-dependent spontaneous mutagenesis was significantly increased in fur mutants. Similarly, SOD deficiency rendered sodA sodB recA mutants nonviable under aerobic conditions. Lethality was suppressed by tonB mutations but not by iron chelation or overexpression of FTN. Thus, superoxide-mediated iron reduction was responsible for oxygen sensitivity. Furthermore, overexpression of SOD partially protected fur recA mutants. We propose that a transient iron overload, which could potentially generate oxidative stress, occurs in wild-type cells on return to normal growth conditions following iron starvation, with the coupling between iron and MnSOD regulation helping the cells cope.
Ustilago maydis secretes ferrichrome-type siderophores, ferric-ion-binding compounds, in response to iron starvation. TA2701, a non-enterobactin-producing, non-ferrichrome-utilizing mutant of Salmonella typhimurium LT-2, was employed as a biological indicator in a novel screening method to isolate three N-methyl-N'-nitro-N-nitrosoguanidine-induced U. maydis mutants defective in the regulation of ferrichrome-type siderophore biosynthesis. These mutants displayed a constitutive phenotype; they produced siderophores in the presence of iron concentrations that would typically repress siderophore synthesis in wild-type strains. A 4.8-kb fragment of U. maydis genomic DNA capable of restoring normal regulation of siderophore biosynthesis in the constitutive mutants was identified. This segment of DNA contains an intronless open reading frame that specifies a protein of 950 amino acids containing two finger motifs similar to those found in the erythroid transcription factor GATA-1. Disruption of this open reading frame in a wild-type strain gave rise to cells that produced siderophores constitutively. Genetic studies indicated that the disruption mutation was allelic to the chemically induced mutations, confirming that the structural gene for a regulator rather than a suppressor gene had been cloned. Northern (RNA) analysis of the gene revealed a 4.2-kb transcript that is expressed constitutively at low levels in wild-type cells. The data support the hypothesis that this gene, which we designate urbs1 (Ustilago regulator of biosynthesis of siderophores), acts directly or indirectly to repress biosynthesis of siderophores in U. maydis.
Catalases are ubiquitous hydrogen peroxide-detoxifying enzymes that are central to the cellular antioxidant response. Of two catalase activities detected in the fungus Aspergillus nidulans, the catA gene encodes the spore-specific catalase A (CatA). Here we characterize a second catalase gene, identified after probing a genomic library with catA, and demonstrate that it encodes catalase B. This gene, designated catB, predicts a 721-amino-acid polypeptide (CatB) showing 78% identity to an Aspergillus fumigatus catalase and 61% identity to Aspergillus niger CatR. Notably, similar levels of identity are found when comparing CatB to Escherichia coli catalase HPII (43%), A. nidulans CatA (40%), and the predicted peptide of a presumed catA homolog from A. fumigatus (38%). In contrast, the last two peptides share a 79% identity. The catalase B activity was barely detectable in asexual spores (conidia), disappeared after germination, and started to accumulate 10 h after spore inoculation, throughout growth and conidiation. The catB mRNA was absent from conidia, and its accumulation correlated with catalase activity, suggesting that catB expression is regulated at the transcription level. In contrast, the high CatA activity found in spores was lost gradually during germination and growth. In addition to its developmental regulation, CatB was induced by H2O2, heat shock, paraquat, or uric acid catabolism but not by osmotic stress. This pattern of regulation and the protective role against H2O2 offered by CatA and CatB, at different stages of the A. nidulans life cycle, suggest that catalase gene redundancy performs the function of satisfying catalase demand at the two different stages of metabolic and genetic regulation represented by growing hyphae versus spores. Alternative H2O2 detoxification pathways in A. nidulans were indicated by the fact that catA/catB double mutants were able to grow in substrates whose catabolism generates H2O2.
The major facilitator superfamily (MFS) is one of the two largest families of membrane transporters found on Earth. It is present ubiquitously in bacteria, archaea, and eukarya and includes members that can function by solute uniport, solute/cation symport, solute/cation antiport and/or solute/solute antiport with inwardly and/or outwardly directed polarity. All homologous MFS protein sequences in the public databases as of January 1997 were identified on the basis of sequence similarity and shown to be homologous. Phylogenetic analyses revealed the occurrence of 17 distinct families within the MFS, each of which generally transports a single class of compounds. Compounds transported by MFS permeases include simple sugars, oligosaccharides, inositols, drugs, amino acids, nucleosides, organophosphate esters, Krebs cycle metabolites, and a large variety of organic and inorganic anions and cations. Protein members of some MFS families are found exclusively in bacteria or in eukaryotes, but others are found in bacteria, archaea, and eukaryotes. All permeases of the MFS possess either 12 or 14 putative or established transmembrane α-helical spanners, and evidence is presented substantiating the proposal that an internal tandem gene duplication event gave rise to a primordial MFS protein prior to divergence of the family members. All 17 families are shown to exhibit the common feature of a well-conserved motif present between transmembrane spanners 2 and 3. The analyses reported serve to characterize one of the largest and most diverse families of transport proteins found in living organisms.
Iron, although essential, is toxic in excess and for this reason the uptake of the element is regulated at the membrane level in all living species. A study of the molecular genetics of the regulatory process may be initiated via use of microorganisms as a model, and the enteric bacterium Escherichia coli K-12 is the species of choice. A particular high affinity iron assimilation complex coded on pCol VK-30 of E. coli has been demonstrated by cloning techniques to consist of five contiguous genes which perform the biosynthesis and transport of the siderophore aerobactin. The operon is preceded upstream by a regulatory element containing major and minor promoters. Quantitative protection experiments with S1 nuclease used in conjunction with lacZ operon and protein fusions proved that gene expression is controlled by iron acting at the transcriptional level. A lacZ operon fusion was used to select mutants, generated with Tn5, displaying constitutive expression of all high affinity iron assimilation components in E. coli. The mutation was mapped at 15.7 min on the chromosome. The mutation was then cloned on pBR322 and the wild-type gene recovered by homologous recombination. The results suggest that in the presence of iron a chromosomal product acting as a repressor shuts down expression of all components of the siderophore pathway which, in turn, restricts further uptake of iron into the cell.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
We investigated the effects of the fermentation with Bacillus natto on the allergenicity ascribed to the major soybean allergen, Gly m Bd 30K. Time dependent decreases in 1) reactivity for IgE antibody from a soybean-sensitive patient and 2) reactivity for a monoclonal antibody were observed during fermentation, and the electrophoretic patterns of the proteins in the fermented soybean showed all the proteins were hydrolyzed to the small fragments with molecular weight below 10,000. These results suggested that the epitopes of the Gly m Bd 30K against the mouse monoclonal IgG and human serum IgE were decomposed by the digestion with the proteases of Bacillus natto during fermentation and that natto might be a hypoallergenic soybean product.
The ARG-11 gene in Saccharomyces cerevisiae encodes a protein with the characteristic features of a family of 35 related membrane proteins that are encoded in the fungal genome. Some of them are known to transport various substrates and products across the inner membranes of mitochondria, but the functions of 29 members of the family are unknown. The yeast ARG-11 protein has been over-produced as inclusion bodies in Escherichia coli. It has been solubilized in the presence of sarkosyl, re-constituted into liposomes and shown to transport ornithine in exchange for protons. Its main physiological role is probably to take ornithine synthesized from glutamate in the mitochondrial matrix to the cytosol where it is converted to arginine.
Aus Kulturen des Actinomyceten-Stammes ETH 28 832 (Streptomyces aureofaciens) wurden die neuen Antibiotica Scopamycin A und B isoliert und charakterisiert. Die Scopamycine sind lipophile Neutralstoffe mit gleichen, charakteristischen UV-Spektren. Scopamycin A und B unterscheiden sich nur im Rf-Wert im Dünnschichtchromatogramm. Die Scopamycine rufen in geringen Konzentrationen starke, Hexenbesen-artige Veränderungen an den Hyphen verschiedener Pilze hervor. Gegen Bakterien zeigen die Scopamycine keine Wirkung.
The major facilitator superfamily (MFS) is one of the two largest families of membrane transporters found on Earth. It is present ubiquitously in bacteria, archaea, and eukarya and includes members that can function by solute uniport, solute/cation symport, solute/cation antiport and/or solute/solute antiport with inwardly and/or outwardly directed polarity. All homologous MFS protein sequences in the public databases as of January 1997 were identified on the basis of sequence similarity and shown to be homologous. Phylogenetic analyses revealed the occurrence of 17 distinct families within the MFS, each of which generally transports a single class of compounds. Compounds transported by MFS permeases include simple sugars, oligosaccharides, inositols, drugs, amino acids, nucleosides, organophosphate esters, Krebs cycle metabolites, and a large variety of organic and inorganic anions and cations. Protein members of some MFS families are found exclusively in bacteria or in eukaryotes, but others are found in bacteria, archaea, and eukaryotes. All permeases of the MFS possess either 12 or 14 putative or established transmembrane alpha-helical spanners, and evidence is presented substantiating the proposal that an internal tandem gene duplication event gave rise to a primordial MFS protein prior to divergence of the family members. All 17 families are shown to exhibit the common feature of a well-conserved motif present between transmembrane spanners 2 and 3. The analyses reported serve to characterize one of the largest and most diverse families of transport proteins found in living organisms.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Extracts of Fusarium roseum (ATCC 12822) contain an enzyme which hydrolyzes the ornithine ester bonds of fusarinine C, a cyclic trihydroxamic acid produced by this organism. The methyl ester of Ndelta-dinitrophenyl-L-ornithine is also a substrate for the enzyme, and an assay was devised using this substrate. The enzyme exhibits a sharp maximum of activity at pH 7.5 and is extremely temperature sensitive. It is strongly inhibited by HgCl2 and p-chloromercuribenzoate, and it is competitively inhibited by Ndelta-dinitrophenyl-D-ornithine methyl ester (Ki = 0.3mM). Methyl esters of glycine, L-alanine, dinitrophenyl-L-alanine, dinitrophenyl-beta-alanine, and Ndelta-dinitrophenyl-Nalpha-acetyl-L-ornithine are not substrates, although Nepsilon-dinitrophenyl-L-lysine methyl ester is as effective as the ornithine derivative. Nonspecific lipases do not hydrolyze ornithine esters, nor does trypsin. The three ester bonds of fusarinine C are progressively hydrolyzed by the enzyme to eventually yield the monomer, fusarinine. The ferric chelate of fusarinine C is not hydrolyzed. An enzyme from Penicillium sp. was isolated with identical properties toward Nbeta-dinitro-phenyl-L-ornithine methyl ester as substrate. It also hydrolyzes N,N',N"-triacetylfusarinine C, a cyclic trihydroxamate containing Nalpha-acetylornithine ester bonds, which is produced by this organism. This substrate is hydrolyzed to Nalpha-acetylfusarine. In contrast to the Fusarium enzyme, this enzyme is fully active toward the ferric trihydroxamate chelate. However, replacement of iron by aluminum leads to a completely inactive substrate. Production of the enzyme is severely suppressed by iron in the growth medium. It is proposed that these specific ornithylesterases provide a mechanism of cellular iron release by hydrolysis of the ferric ionophores, and that an iron-exchange step occurs prior to, and is a prerequisite for, hydrolysis of the ester bonds.
The principal iron uptake system of Saccharomyces cerevisiae utilizes a reductase activity that acts on ferric iron chelates external to the cell. The FRE1 gene product is required for this activity. The deduced amino acid sequence of the FRE1 protein exhibits hydrophobic regions compatible with transmembrane domains and has significant similarity to the sequence of the plasma membrane cytochrome b558 (the X-CGD protein), a critical component of a human phagocyte oxidoreductase, suggesting that FRE1 is a structural component of the yeast ferric reductase. FRE1 mRNA levels are repressed by iron. Fusion of 977 base pairs of FRE1 DNA upstream from the translation start site of an Escherichia coli lacZ reporter gene confers iron-dependent regulation on expression of beta-galactosidase in yeast. An 85-base-pair segment of FRE1 5' noncoding sequence contains a RAP1 binding site and a repeated sequence, TTTTTGCTCAYC; this segment is sufficient to confer iron-repressible transcriptional activity on heterologous downstream promoter elements.
Clinical and environmental isolates of Aspergillus fumigatus synthesized extracellular siderophores when grown in defined medium. Six hydroxamate siderophores were purified from culture filtrates and identified by thin layer chromatography. The most prominent siderophore was identified as N,N',N"-triacetylfusarinine C and the second most prominent siderophore was identified as ferricrocin. In addition, a hydrolytic product of N,N',N"-triacetylfusarinine C was identified. Three other siderophores were present in smaller amounts and were not identified. Since the same siderophores were produced by isolates from diseases of varying severity and from environmental material, it is unlikely that the extracellular siderophores function as virulence factors during infection. However, they may function as growth factors by mediating iron uptake by the fungus in the micro-environment of the inflammatory focus.
The single actin gene from the filamentous fungus Aspergillus nidulans has been isolated and characterized. The only other organism reported to contain just one actin gene is another Ascomycete, the budding yeast Saccharomyces. The nucleotide sequence of the A. nidulans actin gene predicts a polypeptide containing the N-terminal sequence identifying the gamma-actin isotype. Until now this characteristic N terminus has only been reported to occur in vertebrate actin sequences. A monospecific anti-gamma-actin antiserum recognizes a single 42-kDa band in immunoblots of total Aspergillus protein. None of the six introns in the A. nidulans actin gene sequence aligns precisely with those found in other actin genes. One, unlike other known actin introns, is located in the 3'-untranslated region of the gene. The 5' and 3' ends of the gene have been characterized. The Aspergillus actin gene has a heterogeneous transcript size due to the presence of several different 3' termini. Of four characterized polyadenylated transcripts, only the longest contains a typical AATAAA polyadenylation signal near its 3' terminus. Using an integrative plasmid containing Aspergillus actin sequences and the pyr4 gene from Neurospora, the A. nidulans actin gene has been mapped to the first chromosome.
To analyse gene expression signals in Aspergillus we have constructed a set of integration vectors each of which contains in front of the Escherichia coli 'lacZ gene a unique BamHI site in one of the three possible translational reading frames and the A. nidulans argB gene as a selection marker. The vectors allow in-phase translational fusion of any gene to 'lacZ. After transformation of an A. nidulans argB strain, the vectors integrate with a high percentage at the argB locus of the genome, as a single copy. The insertion of the fusion genes at the argB locus assures the constancy of influences of the chromosomal environment on gene expression.
Double radioactive label transport assays with iron, chromium, and gallium chelates were used to investigate the mechanism of iron uptake by Ustilago sphaerogena. In iron-deficient cells, ferrichrome A iron was taken up without appreciable uptake of the ligand. Iron-sufficient cells partially accumulated the ligand with the metal. The chromium- and gallium-containing analogs of ferrichrome A were transported as intact chelates. Ferrichrome A iron uptake was inhibited by dipyridyl. The data suggest that the intact ferrichrome A chelate binds to a specific receptor, the iron is then separated from the ligand at the membrane by reduction, and the metal is released to the inside of the cell while the ligand is released to the exterior. The reduction step is not transport rate limiting. Iron chelated to citrate was taken up by an energy-dependent process. The citrate ligand was not taken up with the metal. Uptake was sensitive to dipyridyl and ferrozine. Chromic ion chelated to citrate was not transported, suggesting that the iron, rather than the chelate, is recognized by the receptor or that reduction of the metal is required for transport.
Aspergillus nidulans and Penicillium chrysogenum produce specific cellular siderophores in addition to the well-known siderophores
of the culture medium. Since this was found previously in Neurospora crassa, it is probably generally true for filamentous
ascomycetes. The cellular siderophore of A. nidulans is ferricrocin; that of P. chrysogenum is ferrichrome. A. nidulans also
contains triacetylfusigen, a siderophore without apparent biological activity. Conidia of both species lose siderophores at
high salt concentrations and become siderophore dependent. This has also been found in N. crassa, where lowering of the water
activity has been shown to be the causal factor. We used an assay procedure based on this dependency to reexamine the extracellular
siderophores of these species. During rapid mycelial growth, both A. nidulans and P. chrysogenum produced two highly active,
unidentified siderophores which were later replaced by a less active or inactive product--coprogen in the case of P. chrysogenum
and triacetylfusigen in the case of A. nidulans. N. crassa secreted coprogen only. Fungal siderophore metabolism is varied
and complex.
The purpose of this article is to explain what oxygen radicals are, how transition metals are involved in their formation and reactivity, and the role played by radicals and metals in some disease states.
The highly active extracellular siderophores previously detected in young cultures of Aspergillus nidulans and Penicillium chrysogenum have been identified as the cyclic ester fusigen (fusarinine C), and its open-chain form, fusigen B (fusarinine B).
Using a scheme for selecting mutants of Saccharomyces cerevisiae with abnormalities of iron metabolism, we have identified a gene, AFT1, that mediates the control of iron uptake. AFT1 encodes a 78 kDa protein with a highly basic amino terminal domain and a glutamine-rich C-terminal domain, reminiscent of transcriptional activators. The protein also contains an amino terminal and a C-terminal region with 10% His residues. A dominant mutant allele of this gene, termed AFT1-1up, results in high levels of ferric reductase and ferrous iron uptake that are not repressed by exogenous iron. The increased iron uptake is associated with enhanced susceptibility to iron toxicity. These effects may be explained by the failure of iron to repress transcription of FRE1, FRE2 and FET3. FRE1 and FRE2 encode plasma membrane ferric reductases, obligatory for ferric iron assimilation, and FET3 encodes a copper-dependent membrane-associated oxidase required for ferrous iron uptake. Conversely, a strain with interruption of the AFT1 gene manifests low ferric reductase and ferrous iron uptake and is susceptible to iron deprivation, because of deficient expression of FRE1 and negligible expression of FRE2 and FET3. Thus, AFT1 functions to activate transcription of target genes in response to iron deprivation and thereby plays a central role in iron homeostasis.
Iron, an essential nutrient, is not readily available in aquatic or terrestrial environments or in animal hosts. Therefore, microbes have developed various strategies for acquiring iron while at the same time protecting themselves from iron's potential toxic effects. The major strategies used by bacteria and fungi to acquire iron include production and utilization of siderophores (ferric specific chelators); utilization of host iron compounds such as heme, transferrin, and lactoferrin; and reduction of Fe(III) to Fe(II) with subsequent transport of Fe(II). Selected examples are discussed with attention to which strategies work best in which environments. The similarities and differences among the different systems with respect to iron binding compounds, receptors, and regulation are also presented.
The syntheses and preliminary biological evaluation of conjugates of a synthetic isocyanurate-based trihydroxamate siderophore with two antifungal agents, 5-FU (conjugate 9) and norneoenactin (conjugate 12), and a macrolide antibiotic, erythromycylamine (conjugate 18), are described. A 19F NMR study was used to determine the hydrolytic stability of conjugate 9 under assay conditions. Preliminary biological studies with ferric complexes of conjugates 9 and 12 indicated that these antifungal agents are recognized by Candida and perhaps are actively transported into the cell by the siderophore-transport mechanisms. While conjugate 18 did not show any significant antibacterial activity, presumably due to size restriction, the 5-FU conjugate 9 appeared to be moderately active against a variety of Gram-positive strains, and was more active than the 5-FC control against some strains of Staphylococcus.
Mutations in arg-13 result in slow growth in minimal medium and can suppress mutations in carbamyl phosphate synthase-aspartate carbamyl transferase within the pyrimidine pathway; the exact biochemical function of the gene product is unknown. To understand the role of arg-13 in arginine metabolism, cosmids rescuing growth in arg-13 mutants were cloned and mapped to the position of arg-13 on LG IR. Northern analysis showed the arg-13 message to contain approximately 2100 nt, although a 1.4-kb genomic fragment truncated at the 5' and 3' ends of the gene encodes a shortened transcript that can rescue arg-13 function. Expression of mRNA arising from the mutant arg-13 gene is induced by arginine starvation, although wild type (arg-13+) is not derepressed in minimal medium. The sequence of the arg-13 gene shows ARG-13 to be a member of the mitochondrial carrier superfamily with three repeats of a approximately 100-amino acid domain, six putative membrane spanning regions, and three copies of the mitochondrial carrier consensus pattern. This information plus available and new nutritional data are consistent with the hypothesis that arg-13 encodes a mitochondrial basic amino acid carrier whose existence was predicted based upon previous physiological, nutritional and biochemical data.
In recent years, significant advances have been made in our understanding of the mechanism and regulation of elemental iron transport in the eukaryote Saccharomyces cerevisiae. This organism employs two distinct iron-transport systems, depending on the bioavailability of the metal. In iron-replete environments, a low-affinity transport system (K(m) = 30 microM) is used to acquire iron. This system may also be used to acquire other metals including cobalt and cadmium. When environmental iron is limiting, a high-affinity (K(m) = 0.15 microM) iron-transport system is induced. Genetic studies in S. cerevisiae have identified multiple genes involved in both iron-transport systems. Cell-surface reductases, FRE1 and FRE2, provide ferrous iron for both systems. A non-ATP-dependent transmembrane transporter (FET4) has been identified as the main component of low-affinity transport. One gene identified to date as part of the high-affinity transport system is FET3, which shows high sequence and functional homology to multicopper oxidases. Accessory genes required for the functioning of this transport system include a plasma-membrane copper transporter (CTR1), an intracellular copper transporter (CCC2), and a putative transcription factor (AFT1). The mechanism by which these genes act in concert to ensure iron accumulation in S. cerevisiae presents an intriguing picture, drawing parallels with observations made in the human system almost 40 years ago.
The general control transcriptional regulator gene cpcA of Aspergillus niger was cloned by complementation of a Saccharomyces cerevisiae delta gcn4 mutant strain. The encoded protein conferred resistance to amino acid analogues when expressed in yeast. Disruption of cpcA in A. niger resulted in a strain which is sensitive towards 3-aminotriazole and fails to respond to amino acid starvation, cpcA encodes a transcript of approximately 2400 nucleotides in length that includes a 5' leader region of 900 nucleotides. The 5' leader region contains two small open reading frames, suggesting translational control of gene expression. Steady-state mRNA levels of cpcA increase by a factor of three upon amino acid starvation. The coding region of cpcA is interrupted by a 57 bp intron and the deduced amino acid sequence displays an approximately 30% overall identity to yeast GCN4p and Neurospora crassa cpc1p. Critical amino acid residues of the transcriptional activation domains of GCN4p are conserved in cpcAp. The basic DNA-binding domain shows up to 70% amino acid sequence identity to other basic zipper (bZIP)-type transcriptional activators. cpcAp binds specifically to a GCN4p recognition element in gel retardation experiments. The C-terminal dimerization domain encodes a leucine zipper with only a single leucine residue.
Employing a PCR-aided strategy, a Penicillium chrysogenum gene (sreP) encoding a putative GATA-transcription factor has been cloned and characterized. Comparison of the genomic and cDNA sequences revealed the presence of an open reading frame (ORF) encoding a protein of 532 amino acids (aa) which is interrupted by two introns. The deduced aa sequence of sreP reveals 50% identity to a regulator of siderophore biosynthesis (URBS1) from Ustilago maydis over a stretch of 200 aa containing two GATA-type zinc finger motifs and a Cys-rich intervening sequence. Northern blot analysis indicated two transcripts of 2.2 and 2.7 kb in approximately equivalent amount. due to two major transcription start sites.
Iron metabolism is regulated in cells to ensure that iron supplies are adequate and nontoxic. The expression of iron metabolism is regulated primarily by posttranscriptional mechanisms. Ferritin, eALAS, SDHb of Drosophila, and mammalian mitochondrial aconitase are translationally regulated. The TfR is regulated at the level of mRNA stability. Iron regulatory proteins are regulated either by assembly or by disassembly of an iron-sulfur cluster (IRP1) or by rapid degradation in the presence of iron (IRP2). The list of targets for IRP-mediated regulation is growing longer, and a range of possibilities for versatile regulation exists, as each IRP can bind to unique targets that differ from the consensus IRE. The reactivity of iron with oxygen and the creation of toxic by-products may be the evolutionary stimulus that produced this system of tight posttranscriptional gene regulation.
Iron is an essential element for nearly all living cells. Thus, the ability of bacteria to utilize iron is a crucial survival mechanism independent of the ecological niche in which the microorganism lives, because iron is scarce both in potential biological hosts, where it is bound by high-affinity iron-binding proteins, and in the environment, where it is present as part of insoluble complex hydroxides. Therefore, pathogens attempting to establish an infection and environmental microorganisms must all be able to utilize the otherwise unavailable iron. One of the strategies to perform this task is the possession of siderophore-mediated iron uptake systems that are capable of scavenging the hoarded iron. This metal is, however, a double-edged sword for the cell because it can catalyze the production of deadly free hydroxyl radicals, which are harmful to the cells. It is therefore imperative for the cell to control the concentration of iron at levels that permit key metabolic steps to occur without becoming a messenger of cell death. Early work identified a repressor, Fur, which as a complex with iron repressed the expression of most iron uptake systems as well as other iron-regulated genes when the iron concentration reached a certain level. However, later work demonstrated that this regulation by Fur was not the only answer under low-iron conditions, there was a need for activation of iron uptake genes as well as siderophore biosynthetic genes. Furthermore, it was also realized that in some instances the actual ferric iron-siderophore complex induced the transcription of the cognate receptor and transport genes. It became evident that control of the expression of iron-regulated genes was more complex than originally envisioned. In this review, I analyze the processes of signal transduction, transcriptional control, and posttranscriptional control of iron-regulated genes as reported for the ferric dicitrate system in Escherichia coli; the pyochelin, pyoverdin, and enterobactin systems in Pseudomonas species; the irgB system in Vibrio cholerae; and the plasmid-mediated anguibactin system in Vibrio anguillarum. I hope that by using these diverse paradigms, I will be able to convey a unifying picture of these mechanism and their importance in the maintenance and prosperity of bacteria within their ecological niches.
The TonB dependent uptake of pyrroloquinoline-1 quinone (PQQ) and secretion of gluconate by Escherichia coli K-12.
Klaus Hantke* and Simon Friz
Summary
Glucose is taken up by Escherichia coli through the phosphotransferase system (PTS) as the preferred carbon source. PTS mutants grow with glucose as a carbon source only in the presence of pyrroloquinoline quinone (PQQ), which is needed as a redox cofactor for the glucose dehydrogenase Gcd. The membrane-anchored Gcd enzyme oxidizes glucose to gluconolactone in the periplasm. For this reaction to occur, external supply of PQQ is required as E. coli is unable to produce PQQ de novo. Growth experiments show that PqqU (YncD) is the TonB-ExbBD dependent transporter for PQQ through the outer membrane. PQQ protected the cells from the PqqU dependent phage IsaakIselin (Bas10) by competition for the receptor protein. As a high affinity uptake system PqqU allows E. coli to activate Gcd even at surrounding PQQ concentrations of about 1 nmol/l. At about 30 fold higher PQQ concentrations the activation of Gcd gets PqqU independent. Due to its small size Pqq may also pass the outer membrane through porins. The PQQ dependent production of gluconate has been demonstrated in many plant growth promoting bacteria that solubilise phosphate minerals in the soil by secreting this acid. Under Pi limiting conditions also E. coli induces the glucose dehydrogenase and secretes gluconate, even in absence of PTS, that is, even when the bacterium is unable to grow on glucose without PQQ.